FIELD OF THE INVENTION

The present invention generally concerns single cell analysis and similar methods for analyzing particles in microscale. Especially, a system and method for moving a particle of interest in a microfluidic channel is provided, wherein a second particle is used to control the movement of the particle of interest. The second particle is capable of being actuated, which initiates movement of the particle of interest.

BACKGROUND OF THE INVENTION

The fast development of single-cell sequencing technologies has led to a higher degree of resolution regarding the characterization of heterogeneous and complex cell populations (see Junker, J.P. & van Oudenaarden, A. (2014) Cell 157: 8-11). Parallel technical advances for single-cell isolation technologies such as fluorescence activated cell sorting (FACS), micromanipulation and microfluidics have further enabled the linkage between the optical analysis of cellular phenotype, such as immunofluorescence staining (IF) with transcriptional profiles via next-generation sequencing (NGS) (see Saliba, A.-E. et al. (2014) Nucleic Acids Research 42(14): 8845-8860). The combination of the optical analysis of cellular phenotype with single-cell sequencing approaches has provided important insights into the transcriptional heterogeneity of pluripotent stem cells, tumour cells and immune cells.

Because current single-cell isolation platforms rely on single time point analysis, they can only provide an instantaneous snapshot of dynamic, cellular phenotypes to link to a transcriptional signature. This makes it impossible to investigate the mechanisms for generating heterogeneities over time. In addition, it is not possible to link transcriptional profiles to the cell function. The understanding of the mechanisms for generating heterogeneities over time is one of the key challenges to get a comprehensive understanding of asynchronous, time-resolved biological processes. For instance, a characteristic of tumorigenesis is the ability of single cells to generate diverse progeny with different potencies. However, the mechanism by which this diversity is generated from a single founding cell remains a highly controversial topic. Resolving the relative contributions of various models of cancer progression, as well as generally defining the mechanisms by which a single cell gives rise to distinctly different progeny in a tumour, requires a means of directly tracking single-cell lineage while making sensitive and comprehensive measurements of end-point cell phenotypes by linking the cell history to end-point transcriptional profiles.

For cell-retrieval after cultivation, major technical hurdles exist with 2D cultivation in current devices. These hurdles include immobilization of non-adherent cells such as cells derived from the hematopoietic system to prevent cell loss during medium exchange and detaching adherent cells from the device for downstream sequencing analysis. Both procedures can substantially alter the inherent cell phenotype and are therefore unsuitable for coupling time- resolved functional phenotypes to the underlying genotype (see Chen, S. et al. (2015) Journal of Immunological Methods 426: 56-61; Badur, M.G. et al. (2015) Biotechnology Journal 10: 1600-1611).

For the analysis of the cell mechanisms it is crucial to cultivate the cells within physiological 3D microenvironments. Performing experiments in desired 3D cell culture micro environments possesses another additional challenge. The physiological relevance of a 3D microenvironment has been extensively studied. Recent studies show that especially for clinically relevant processes, 2D cell culture or droplet culture systems have limitations as they result in abnormal phenotypes (see Hasani-Sadrabadi, M.M. et al. (2020) Materials Horizons 7: 3028-3033).

Recent developments in microfluidic technology have enabled new devices of trapping and culturing single cells and cell pairs (see Gomez-Sjoberg, R. etal. (2007) Analytical Chemistry 79(22): 8557-8563; Tan, W.-H. & Takeuchi, S. (2007) PNAS 104(4): 1146-4451). When combined with traditional microscopes, these systems provide a robust means of analysing functional phenotypes over time but require extensive technical equipment and expansive and complex production processes for cell retrieval to couple functional phenotypes to the underlying genotype. All devices have in common that the components necessary for the cell-retrieval are part of the microfluidic chip. This leads to high production cost and limits the retrieval process to only a few positions. In Gomez-Sjoberg, R. et al. (2007) the retrieval process is based on cell incompatible aluminium patterns on glass substrate which have to be produced by expensive lithography processes. In addition, the retrieval is limited to cell encapsulated in micro-droplets, limiting the duration of the cultivation period due to limited access to nutrition. In Mulas, C. et al. (2020) Lab on a Chip 20: 2580-2591, the cell retrieval was shown with solid cell-laden hydrogel beads. But the retrieval process is based on microfluidic quake valves which have to be produced by a complex multi-layer microfluidic chip design. In addition, the quake valve requires expansive macro valve to be actuated. Another drawback of using quake valves is their large footprint which significantly limits the number of retrievable hydrogel beads.

In Valihrach, L. et al. (2018) International Journal of Molecular Sciences 19: 807-826, the cell-retrieval process is limited to adhesion cells because the cells have to adhere to a substrate which is isolated by a magnetic field. Therefore, this device is not suitable to investigate cells of the hematopoietic system. Another drawback of this technology is the incompatibility to 3D cell culture.

Other microfluidic devices which enable the efficient preparation of cDNA libraries from single-cells for transcriptional analysis (Fluidigm C1 platform) lack the long-term culture and phenotypic time-lapse imaging capabilities to link these transcriptional analyses with functional information. In addition, the error-free handling on this platform depends on the cell phenotype because changes regarding the cell-size significantly influence the flow characteristics in the microfluidic chip. Another disadvantage of those platforms is the incompatibility to 3D cell culture.

The invention aims at avoiding drawbacks of the prior art methods. In particular, it is an object to be able to analyse the functional phenotype of cells within physiological microenvironments by using traditional imaging approaches and link the functional phenotype of a cell to its downstream gene expression profile and genotype. It is another object to be able to perform dynamic studies of living single cells and small populations of cells which can increase the understanding of the interconnecting molecular events coupling phenotypic events to the underlying genotype of particular cells. It is another object to provide a microenvironment to the cells that mimics the conditions the cells encounter in vivo. It is another object to be able to position the spherical hydrogel bead in close proximity to another spherical hydrogel bead which acts as a retrieval bead.

SUMMARY OF THE INVENTION

The present inventors have developed a system for controlling the positioning and movement of a particle for microanalysis. In the system, the means for controlling the movement is separated from the actual microparticle of interest, i.e. the payload particle which carries, e.g., a cell to be analyzed. Said means for movement control is provided with a second microparticle, i.e. the positioning particle, which can be actuated and thereby controls the movement of the payload particle. By separating the two functions - the carrier function of hosting a target product to be analyzed, and the movement and positioning control - manufacturing of the particles is much easier and cost effective. Always the same positioning particles can be used for a microfluidic system, which hence can be produced and distributed in bulk. Furthermore, the payload particles, which are replaced if another target product is analyzed, do not additionally have to comprise movement control means such as magnetic nanoparticles or the like. Furthermore, the means for movement control are not in close contact with the target product to be analyzed and therefore, do not influence the analysis. For example, contact with magnetic nanoparticles or irradiation with light may influence the behavior and reactions of the cells of interest.

In order to increase the ability of the positioning particle to control the movement of the payload particle, two positioning particles may be used which are brought into position as single particles, but then are crosslinked to improve their handling and better secure them in their desired position.

In addition, one significant disadvantage of the prior art methods is the necessity of valves on the microfluidic chip. The integration of microfluidic valves significantly increases the footprint of the microfluidic geometry and thereby limits the multiplexing capacity. The present invention, on the other hand, does not require the use of microfluidic valves because movement control is achieved by a positioning particle. Therefore, this technology is optimally suited for a high degree of multiplexing.

In view of the above, the present invention provides according to a first aspect a system, in particular a system for controlling the positioning and movement of a particle for microanalysis, comprising a microfluidic channel and positioned within said microfluidic channel a payload particle and a positioning particle; wherein the positioning particle is capable of being actuated; wherein actuating the positioning particle initiates movement of the payload particle.

According to a second aspect, the present invention provides a method for moving a payload particle in a microfluidic channel, comprising the steps of

(i) providing a payload particle and a positioning particle in a microfluidic channel;

(ii) initiating movement of the payload particle by actuating the positioning particle.

According to a third aspect, the present invention provides a kit of parts, comprising a payload particle and a positioning particle; wherein the payload particle and the positioning particle are for use in a microfluidic channel; wherein the positioning particle is capable of being actuated; and wherein actuating the positioning particle initiates movement of the payload particle.

According to a fourth aspect, the present invention provides the use of a positioning particle for initiating movement of a payload particle in a microfluidic channel; wherein the positioning particle is capable of being actuated; and wherein actuating the positioning particle initiates movement of the payload particle.

In certain aspects of the invention, at least two positioning particles are used, which are crosslinked with each other once they are in place within the microfluidic channel.

In addition to the above, the present inventors have developed a system for analyzing molecules secreted by single cells or cell pairs. The cell(s) are encapsulated in a payload particle and a capture particle carrying specific ligands for capturing the molecules to be analyzed (analytes) is positioned adjacent to the payload particle. In order to determine background presence of the analyte, a second capture particle is used which is positioned adjacent to the first capture particle, but on the opposite side compared to the payload particle. Using this setup of payload particle and two capture particles in a row, also multiple of these particle groups can be positioned within one system and even within one microfluidic channel. Analytes washed from one payload particle to the next with the microfluidic flow or analytes diffusing from one to the next particle group are accounted for by the second capture particle. Furthermore, the second capture particle can serve as proof that no analytes diffused or were washed from one particle group to the next.

Therefore, the present invention provides according to a fifth aspect a system, in particular a system for analyzing molecules secreted by a payload of interest, comprising a microfluidic channel comprising an inlet and an outlet, and positioned within said microfluidic channel a payload particle comprising a payload of interest, a first capture particle and a second capture particle; wherein the capture particles are capable of capturing analytes released from the payload of the payload particle; and wherein the payload particle and the capture particles are positioned within the microfluidic channel in the direction of from inlet to the outlet in the order of

(i) payload particle,

(ii) first capture particle, and

(iii) second capture particle.

According to a sixth aspect, the present invention provides a method for detecting analytes released by a payload, comprising the steps of

(i) providing a system according to the fifth aspect of the invention;

(ii) incubating the payload of interest in the system for a desired time under conditions at which the payload of interest may release analytes; (iii) determining the amount of analytes captured by the first capture particle and determining the amount of analytes captured by the second capture particle;

(iv) calculating the amount of analytes released by the payload of interest based on the amount of analytes captured by the first and second capture particles as determined in step (iii), wherein the amount of analytes captured by the second capture particle serves as background signal.

In certain embodiments, the payload of interest is one or more cells.

In certain aspects, the system according to the first aspect and the system according to the fifth aspect are combined. In these aspects, the system comprises a payload particle, a first capture particle and a second capture particle, wherein the first and/or the second capture particle additionally also is a positioning particle, or the system further comprises a positioning particle.

Other objects, features, advantages and aspects of the present application will become apparent to those skilled in the art from the following description and appended claims. DETAILED DESCRIPTION OF THE INVENTION

The present invention provides new means for controlling the movement of a particle of interest, the payload particle, in a microfluidic device by using a second particle, the positioning particle, which is controlled via external forces, such as a magnetic field or light. By moving, swelling or shrinking the positioning particle, movement of the payload particle is initiated. For example, the payload particle may be pushed or pulled by positioning particle which is actively moved through movement of a magnetic field. Or the payload particle may be pushed by swelling the positioning particle directly adjacent to the payload particle. Or the positioning particle may block the flow through the microfluidic channel harboring the payload particle, and shrinking the positioning particle enables the flow to reach and move the payload particle. Using this system, only the positioning particle is manipulated, e.g. by swelling or shrinking upon irradiation with light, and only the positioning particle needs to be responsive to the control mechanism, e.g. by comprising magnetic nanoparticles. Thereby, the payload particle comprising the product of interest is not affected by any of these control mechanisms, which therefore do not disturb the analysis. In order to increase the positioning particle's ability to control the movement of the payload particle, and to better secure the positioning particles at their desired position, at least two positioning particles may be used instead of only one positioning particle. The at least two positioning particles may be crosslinked with each other once they are in place. This is achieved by introducing the positioning particles into a microfluidic system as single particles, and crosslinking two adjacent positioning particles once they are located at their desired position.

The present invention further provides new means for detecting analytes released by a payload of interest in a microfluidic device by using a payload particle harboring the payload of interest, e.g. a cell, and a first capture particle and a second capture particle. The first capture particles captures the analytes released by the payload and the second capture particle captures the analytes present in the system background. The system background includes analytes which were released by payloads of other payload particles, especially payload particles present upstream or downstream in the same microfluidic channel, and impurities present in the system. In addition, the second capture particle also accounts for systematic measurement errors in the determination of the amount of analytes captured by the capture particles.

The microfluidic system for controlling positioning and movement of a particle

According to a first aspect, the present invention provides a system comprising a microfluidic channel and positioned within said microfluidic channel a payload particle and a positioning particle; wherein the positioning particle is capable of being actuated; wherein actuating the positioning particle initiates movement of the payload particle.

The system in particular is a microfluidic system. A microfluidic system is a system comprising one or more channels for transport of a fluid, wherein the diameter of the channels is in the sub-millimeter range. In certain embodiments, the microfluidic channel(s) has a diameter in the range of from 1 to 500 pm, preferably from 30 to 200 pm, more preferably from 50 to 120 pm. Specifically, the microfluidic channel(s) may have a diameter of about 70 to 100 pm. The diameter of a microfluidic channel in general refers to the smallest diameter in case breadth and height of the channel are not the same. For example, the microfluidic channel(s) may have a breadth of about 100 pm and a height of about 80 pm. In specific embodiments, the breadth and/or the height of the microfluidic channel is about as large as the diameter of the payload particle and/or the positioning particle and/or the capture particles, if present.

The system may comprise a means for applying a microfluidic flow through the microfluidic channel(s), such as for example a micropump or a defined pressure gradient. Alternatively, a microfluidic flow may be achieved using capillary forces. In certain embodiments, the microfluidic channel is part of a microfluidic chip. In specific embodiments, the microfluidic flow is in the direction from the inlet to the outlet of the microfluidic channel. The system comprises a payload particle and a positioning particle within the microfluidic channel. The system may comprise more than one payload particle and/or more than one positioning particle. The multiple payload particles and multiple positioning particles may be present in the same and/or in different microfluidic channels of the system. In preferred embodiments, one payload particle and one positioning particle form a pair, wherein actuating the positioning particle initiates movement of the paired payload particle. In the following, positioning particle and payload particle especially refer to the particles of a pair of positioning and payload particle.

In certain embodiments, the system comprises a plurality of pairs positioned within said microfluidic channel, wherein each pair comprising exactly one payload particle and one positioning particle. Especially, a positioning particle of a selected pair is capable of being actuated without actuating positioning particles of other pairs. Actuating the positioning particle of a selected pair initiates movement of the payload particle of said selected pair.

In certain embodiments, the positioning particle and the payload particle are adjacent to each other or in the vicinity of each other in the microfluidic channel. In particular, the positioning particle and the payload particle are positioned within the microfluidic channel at a distance of 200 pm or less, preferably 100 pm or less, and more preferably 20 pm or less. Most preferably, the positioning particle and the payload particle are in contact with each other.

In the system, the positioning particle is capable of being actuated, and actuating the positioning particle initiates movement of the payload particle. "Actuating" as used herein especially means that a force is applied to the positioning particle and the positioning particle reacts to said force. The force in particular may be a magnetic field or light. In preferred embodiments, the force is not the microfluidic flow within the microfluidic channel or system. In particular, the force is applied from outside of the microfluidic channel. Hence, in preferred embodiments, the positioning particle is not actuated by a microfluidic flow.

As long as the positioning particle is not actuated, it is in a resting state. In the resting state, the positioning particle does not initiate movement of the payload particle. As long as the positioning particle does not initiate movement of the payload particle, the payload particle is in a resting state. In specific embodiments, the positioning particle it its resting state prevents the payload particle from moving. In certain embodiments, the positioning particle in its resting state blocks or significantly reduces a microfluidic flow through the microfluidic channel and/or a section of the microfluidic channel. A significant reduction of the microfluidic flow for example is a reduction by at least 25%, preferably at least 50%, more preferably at least 75%.

In certain embodiments, the positioning particle in its resting state is fixed at its position in the microfluidic channel. In particular, the positioning particle is wedged in the microfluidic channel due to its size. Especially, the positioning particle in its resting state is not moved by a microfluidic flow applied to the system or the microfluidic channel. In certain embodiments, the payload particle in its resting state is fixed at its position in the microfluidic channel. In particular, the payload particle is wedged in the microfluidic channel due to its size. In specific embodiments, the positioning particle and/or the payload particle are fixed at specific positions in the microfluidic channel. These positions for example have a smaller diameter than other parts of the microfluidic channel or are surrounded by parts of the microfluidic channel with smaller diameters. Due to such designs, a force has to be applied to the positioning particle and/or the payload particle in order to move them from their position. In certain embodiments, these specific positions are positions within a microfluidic positioning device. Suitable designs of the microfluidic channel are described, for example in DE 10 2020004660.6 and WO 2022/023524 A1.

In specific embodiments, the microfluidic channel comprises a positioning device for sequential positioning of particles, wherein the positioning device has a preferably rigid delimitation structure, wherein the delimitation structure forms a first receptacle (23) for positioning a particle and a second receptacle (24) for positioning a particle, wherein the first receptacle (23) and the second receptacle (24) are arranged in series, wherein the positioning device has a device opening (25), through which fluid can flow into the positioning device, wherein a device channel extends from the device opening (25) into the positioning device, wherein the positioning device (21) has at least one bypass channel (26, 27), wherein the positioning device (21) has a branch point (28), wherein the device channel and the bypass channel (26, 27) are branched via the branch point (28) in such a way that a particle located in the branch point (28) can flow into the bypass channel (26, 27) or via the device opening (25) into the device channel. The device channel has a greater hydrodynamic resistance than the bypass channel (26, 27). Alternatively, the device channel has the same hydrodynamic resistance as the bypass channel (26, 27). The terms “positioning device” and “bead trap” are used as synonyms herein.

The positioning device may in certain embodiments comprise a first confining portion (29) forming a first opening (25) - preferably being the device opening (25) - such that a particle can pass through the first opening (25) into the first receptacle (23), and a second confining portion (30) forming a second opening (31) such that a particle can pass through the second opening (31) into the second receptacle (24).

In certain embodiments, the first confining portion (29) and the second confining portion (30) form the first receptacle (23), wherein the positioning device comprises a third confining portion (32), wherein the second confining portion (30) and the third confining portion (32) form the second receptacle (24). The first confining portion (29) and the second confining portion (30) may in particular each comprises two separate parts which define the first opening (25) and the second opening (31), respectively. In particular, the positioning device is configured such that a particle (41) located entirely in the first receptacle (23) is prevented from moving through the first opening (25) and from moving toward the second opening (31) because the particle has a diameter large enough that it cannot pass through either the first opening (25) or the second opening (31) and contacts the first confining portion (29) and the second confining portion (30), and a particle (40) located entirely in the second receptacle (24) is prevented from moving through the second opening (31) and from moving in the opposite direction because the particle has a diameter large enough that it cannot pass through either the second opening (31) and contacts the second confining portion (30) and the third confining portion (32).

In specific embodiments, the positioning particle and the payload particle are present in the positioning device. In particular, the payload particle (41) is located in the first receptacle (23) and the positioning particle (40) is located in the second receptacle (24). Upon actuating of the positioning particle, movement of the payload particle through the first opening (25) out of the first receptacle (23) may be initiated. In particular, the payload particle is moved from the first receptacle (23) to the branching point (28), where it may be transported away through the bypass channel (26, 27) by a microfluidic flow applied to the microfluidic channel.

In certain embodiments, the delimitation structure of the positioning device additionally forms a third receptacle for positioning a particle which is arranged in series with the first and second receptacles after the second receptacle. In these embodiments, the third confining portion forms a third opening such that a particle can pass through the third opening into the third receptacle. The third confining portion may in these embodiments comprise two separate parts which define the third opening. The positioning device may comprise a fourth confining portion, wherein the third confining portion and the fourth confining portion form the third receptacle. In particular, in these embodiments a particle located entirely in the third receptacle is prevented from moving through the third opening and from moving in the opposite direction because the particle has a diameter large enough that it cannot pass through either the third opening and contacts the third confining portion and the fourth confining portion.

The positioning particle may be located in front of or behind the payload particle in the direction of the microfluidic flow in the microfluidic channel. In certain embodiments, the movement of the payload particle which is initiated is in the direction of the microfluidic flow. In alternative embodiments, the movement of the payload particle which is initiated is against the direction of the microfluidic flow. In other embodiments, no microfluidic flow is applied to the microfluidic channel.

The positioning particle and the payload particle in particular are not bound or attached or attracted to each other. Thus, in certain embodiments the positioning particle does not bind to the payload particle via covalent and/or ionic bonds, via magnetic attraction, via ligand interaction, and/or via structural interaction. In specific embodiments, the positioning particle is not coupled to the payload particle. Initiation of movement of the payload particle via actuating the positioning particle hence is in particular effected via pushing of the payload particle or movement of the fluid within the microfluidic channel.

In certain embodiments, the system comprises at least two positioning particles within said microfluidic channel, wherein the positioning particles are crosslinked with each other and are capable of being actuated; wherein actuating the positioning particles initiates movement of the payload particle. In these embodiments, the payload particle is in contact with at least one of the positioning particles, wherein especially the payload particle is contacted by one positioning particle and this positioning particle is further crosslinked to the other positioning particle. Features and embodiments described herein for a positioning particle likewise also apply to two or more positioning particles which are crosslinked with each other.

The positioning particle

In the system, the positioning particle is used for controlling the movement and position of the payload particle. The positioning particle is capable of being actuated. In particular, a force may be applied to the positioning particle and the positioning particle reacts to the force. The force is initiated from outside of the microfluidic channel. Suitable forces include, for example, magnetic fields or irradiation with light, and suitable reactions of the positioning particle include, for example, movement within the microfluidic channel, shrinkage, swelling, and production of gas. A microfluidic flow applied to the system or microfluidic channel or the momentum induced by such a microfluidic flow is not a force for actuating the positioning particle in the sense of the present invention.

Actuating the positioning particle initiates movement of the payload particle. Especially, the reaction of the positioning particle to the external force leads to a movement of the payload particle. For example, the payload particle may be pushed or pulled by the positioning particle, either directly through direct contact of both particles, or indirectly through another particle or through undertow or thrust of the fluid within the microfluidic channel, or the positioning particle may allow flow of the fluid in the microfluidic channel when actuated.

In embodiments wherein the system comprises at least two positioning particles, the at least two positioning particles may be actuated simultaneously and in particular show the same reaction to being actuated. For example, the at least two positioning particles move in the same direction when applying a magnetic field.

The at least two positioning particles in the microfluidic channel are crosslinked with each other. The positioning particles in particular are chemically crosslinked with each other. In certain embodiments, the at least two positioning particles contain crosslinking agents on their surface which chemically react with each other to form stable covalent bonds when activated. Activation of the crosslinking agents may be achieved by applying an activating agent to the microfluidic channel comprising the at least two positioning particles. In a particular embodiment, the at least two positioning particles may be crosslinked by applying light, in particular UV-light to the positioning particles. The light may in particular catalyze a radical photopolymerization of crosslinking agents such as (meth)acrylic acid moieties.

In a particular embodiment, the crosslinking agents comprise a degradable protection group, preferably degradable by change of the pH-value (e.g. a protection group comprising a hydrazone moiety for acidic degradation), by action of an enzyme (e.g. a protection group comprising a peptide as a target site for enzymatically degradation such as hydrolysis), by action of reducing agents (e.g. a protection group comprising a disulfide-moiety for degradation by glutathione and DTT), by action of oxidizing agents (e.g. a protection group comprisign vicinal diols for degradation by periodate oxidation), by action of miscellaneous chemical agents (e.g. a protection group comprising a thioether moiety for proteolytic degradation), by action of electromagnetic waves such as UV light (e.g. a protection group sensitive to UV irradiation such as nitrobenzyl).

Suitable examples of crosslinking reactions are selected from: (i) a covalent bond formation, preferably selected from (aa) enzymatically catalyzed reactions, such as reactions catalyzed with transglutaminase factor Xllla, (bb) not-enzymatically catalyzed reactions, such as click chemistry or photo-catalyzed reactions and/or (cc) uncatalyzed reactions, such as copper- free highly selective click chemistry, Michael-type addition or Diels-Alder conjugation; (ii) non-covalent bond formation, preferably selected from (aa) hydrogen bonds, preferably formed by nucleic acids or nucleic acid analogs, (bb) hydrophobic interactions, (cc) Van-der- Waals interactions and (dd) electrostatic interactions; and (iii) combinations of the foregoing. Suitable crosslinking agents for the crosslinking reaction are carboxyl, thiol and amine groups, peptides, nucleic acids or unsaturated imides. Suitable activating agents for initiating the crosslinking reaction include acids, alkaline solution, enzymes, reducing agents, oxidizing agents, miscellaneous chemical agents, electromagnetic waves such as UV light, carboxy-, thiol-, amine-, or unsaturated imide-functionalized components, preferably polyethylene glycol (PEG) such as poly(ethylene glycol) bis(amine) or poly(ethylene glycol) dithiol or di(N- succinimidyl) functionalized components or dithiol moieties such as di-thiodipropionic acid di(N-hydroxysuccinimide ester or carboxyfunctionalized disulfides such as 2-carboxyethyl disulfide or hetero-bifunctional reactive components such as 3-(maleimido)-propionic acid N- hydroxysuccinimide ester.

Magnetically responsive positioning particles

In specific embodiments, the positioning particle is responsive to a magnetic field. In these embodiments, actuating the positioning particle in particular includes moving the positioning particle within the microfluidic channel using a magnetic field. The movement of the positioning particle in particular moves the payload particle.

In certain embodiments, the positioning particle is moved towards the payload particle. In these embodiments, the payload particle is pushed in the direction of the movement of the positioning particle. This may be achieved either by direct contact to the positioning particle, or by the increased pressure in the fluid between the payload particle and the positioning particle caused by the movement of the positioning particle. In embodiments where the microfluidic channel comprises a positioning device and the payload particle is located in the first receptacle and the positioning particle is located in the second receptacle, the positioning particle may be moved towards the payload particle from the second receptacle to the first receptacle, thereby pushing the payload particle out of the first receptacle.

In other embodiments, the positioning particle is moved away from the payload particle. In these embodiments, the payload particle is moved by the undertow created by the movement of the positioning particle, and/or by a microfluidic flow applied to the microfluidic channel and/or a change of microfluidic flow that is initiated due to the actuation of the positioning particle.

For movement of the positioning particle using a magnetic field, the positioning particle in particular is responsive to a magnetic field because it comprises magnetic material. In certain embodiments, the positioning particle comprises magnetic nanoparticles. The magnetic material, especially the magnetic nanoparticles, may be ferromagnetic, ferrimagnetic, paramagnetic, superparamagnetic, or diamagnetic. In certain embodiments, the magnetic material, especially the magnetic nanoparticles, has a high uniaxial magnetocrystalline anisotropy.

In certain embodiments, the magnetic material, especially the magnetic nanoparticles, comprise material selected from FesCU, Nd, Ni, Co, NdaFenB, and tetracyanoquinodimethane, or a combination thereof. Specifically, the magnetic material, especially the magnetic nanoparticles, may consist of such material. In specific embodiments, the magnetic material, especially the magnetic nanoparticles, is coated, for example with polyaniline.

In specific embodiments where the positioning particle is responsive to a magnetic field, the system further comprises a magnet as source of the magnetic field. Exemplary magnets include permanent magnets and electromagnets. For example, the source of the magnetic field may be a neodymium magnet. In certain embodiments, the system further comprises a magnetizable needle. This needle is magnetized by the source of the magnetic field and can be used to specifically target the magnetic field to the positioning particle. The tip of the needle may especially be at a distance in the range of from 1 to 2000 pm from the positioning particle, preferably from 20 to 1500 pm, more preferably from 100 to 500 pm. In particular, the magnetizable needle and the distance of its tip to the positioning particle are designed so that one specific positioning particle within the system may be actuated while other positioning particles in the system are not actuated or affected.

For actuating the positioning particle, the source of the magnetic field can be moved relative to the microfluidic channel and/or turned on and off. For example, the microfluidic channel is fixed at its position and the source of the magnetic field is moved, or the source of the magnetic field is fixed at its position and the microfluidic channel is moved, or both the microfluidic channel and the source of the magnetic field are moved. In this respect, source of the magnetic field refers to the magnet as well as to any magnetizable material used for actuating the positioning particle, such as the magnetizable needle. In a specific embodiment, the source of the magnetic field is fixed in its position and the microfluidic channel is moved in order to change the position of the positioning particle within the microfluidic channel. In a further embodiment, the microfluidic channel is moved to locate the source of the magnetic field at a desired position of the microfluidic channel, especially in the X and Y axis, and the source of the magnetic field is moved towards or away from the microfluidic channel, especially in the Z axis, so that the magnetic field gets into or out of range to actuate the positioning particle. In this embodiment, the microfluidic channel and optionally also the microfluidic chip in particular extends in the X and/or Y axis.

In certain embodiments, the source of the magnetic field, in particular the magnetizable needle, is attached to a device for monitoring the microfluidic channel. In particular, the device for monitoring the microfluidic channel is an optical device, such as an objective which may be coupled to a camera or a microscope, especially an objective of a camera. In these embodiments, operating the source of the magnetic field, actuating of the positioning particle and movement of the payload particle can directly visually be monitored. Thus, in a specific embodiment, the system additionally comprises an optical device for monitoring the microfluidic channel and a source of a magnetic field, in particular a magnetizable needle, attached to the optical device. In particular, the source of the magnetic field is attached to the optical device in such a manner that the part of the source of the magnetic field which is used for actuating the positioning particle, such as the tip of a magnetizable needle, is within the field of view of the optical device.

In certain embodiments, the system comprises two optical devices, a first optical device to which the source of the magnetic field is attached, and a second optical device without the source of the magnetic field. The first optical devise is for monitoring the microfluidic channel during actuating of the positioning particle and initiation of the movement of the payload particle, and the second optical device is for monitoring the microfluidic channel when actuating the positioning particle is not desired. The two optical devices preferably can switch positions so that the same part of the microfluidic channel can be monitored after switching of the optical devices. Light-responsive positioning particles

In specific embodiments, the positioning particle is responsive to light. In these embodiments, actuating the positioning particle in particular includes applying light to the positioning particle. The positioning particle responds to the irradiation with light, for example by swelling, shrinking or releasing gas.

In certain embodiments, the light causes the positioning particle to shrink. Shrinking of the positioning particle in particular allows a microfluidic flow applied to the microfluidic channel to pass and/or move the positioning particle. In particular, shrinking of the positioning particle induces a local change of hydrodynamic resistance and thus, a change in the microfluidic flow. Thereby also the payload particle present in the same microfluidic channel is moved, especially due to the change of the microfluidic flow caused by actuation of the positioning particle or by the positioning particle pushing the payload particle. In particular, the positioning particle in a resting state blocks microfluidic flow through the microfluidic channel. Thereby, the payload particle is not affected by a flow and rests in its position. Upon irradiation of the positioning particle, it shrinks and does no longer block flow through the microfluidic channel. In consequence, the flow reaches the payload particle and moves it through the microfluidic channel. Alternatively, the positioning particle may be wedged in the microfluidic channel without completely blocking microfluidic flow through the channel. Thereby, the positioning particle is fixed in its position and blocks the path for the payload particle. Upon irradiation and shrinking, the positioning particle is no longer wedged and both the positioning particle and the payload particle are carried away by the microfluidic flow.

Shrinking of the positioning particle may be achieved, for example, by generation of complementary charged chemical groups upon irradiation with light. In particular, the light induces hydrolysis, protonation or deprotonation of chemical groups within the material of the positioning particle. Thereby, complementary charged chemical groups are generated, which decreases electrostatic repulsion between charged groups and/or decreases osmotic pressure.

In certain embodiments, the light causes the positioning particle to swell. Swelling of the positioning particle in particular pushes the payload particle away from the positioning particle, either by direct contact to the positioning particle, or by the increased pressure in the fluid between the payload particle and the positioning particle caused by the swelling of the positioning particle. In particular, the positioning particle is in direct contact to the payload particle and upon irradiation and swelling, the positioning particle pushes the payload particle out of its resting position. In embodiments where the microfluidic channel comprises a positioning device and the payload particle is located in the first receptacle and the positioning particle is located in the second receptacle, the positioning particle may swell into the first receptacle, thereby pushing the payload particle out of the first receptacle. Swelling of the positioning particle may be achieved, for example, by generation of similar charged chemical groups upon irradiation with light. In particular, the light induces hydrolysis, protonation or deprotonation of chemical groups within the material of the positioning particle. Thereby, chemical groups with the same charge are generated, which increases electrostatic repulsion between charged groups and/or increases osmotic pressure.

In certain embodiments, the light causes the positioning particle to release gas. The gas forms a bubble in the microfluidic channel. Formation of the bubble pushes the payload particle out of its resting position. The bubble may form between the positioning particle and the payload particle, pushing the payload particle away from the positioning particle, or it may form at the side of the positioning particle facing away from the payload particle, pushing both the positioning particle and the payload particle into the same direction. In specific embodiments, the formed bubble has a diameter in the range of from 1 to 500 pm, preferably from 1 to 90 pm.

Applying light to the positioning particle may in particular cause a local change of characteristics of the positioning particle. Especially, the pH value, the temperature, the redox potential, the ionic charge, and/or the intermolecular bond formation such as van der Waals, hydrogen bond and ionic interactions may be changed upon irradiation with light. The positioning particle may in particular comprise one or more of the following group of suitable materials:

• Poly(N-isopropylacrylamide)

• Poly(N-isopropylmethacrylamide)

• Poly(acrylic acid-co-acrylamide)

• Polyacrylamide

• Poly(N,N-diethylacrylamide)

• Poly(N,N-dimethylaminoethyl methacrylate)

• Poly(ethylene glycol)

• Dibenzaldehyde-terminated poly(ethylene glycol)

• Poly(methyl vinyl ether)

• Poly(vinyl alcohol)

• Poly(N-vinylcaprolactam)

• Poly(vinylpyrrolidone)

• Spiropyran derivates

The light applied to the positioning particle in particular comprises wavelengths in the range from 1 nm to 10 cm, preferably from 100 nm to 1000 nm, more preferably 365 nm to 900 nm.

In specific embodiments where the positioning particle is responsive to light, the system further comprises a light source. The light source may be any light source known in the art suitable for illuminating the positioning particle. Especially, the light source is capable of specifically illuminating the positioning particle. In particular, the light source preferably is designed so that one specific positioning particle within the system may be actuated while other positioning particles in the system are not actuated or affected. Exemplary light sources include a laser, especially a laser with a small spot size which is smaller than the diameter of the particles of the system. A suitable spot size of the laser is for example in the range of 0.1 to 50 pm, preferably 1 to 10 pm, such as about 3 pm.

For actuating the positioning particle, the light source can be moved relative to the microfluidic channel and/or turned on and off. For example, either the microfluidic channel is fixed at its position and the light source is moved, or the light source is fixed at its position and the microfluidic channel is moved. In this respect, light source refers to device actually producing the light as well as to any devices used for directing the light to the positioning particle, such as fiber optic devices.

The payload particle

The payload particle may be any suitable particle for use in microfluidic systems. In particular, the payload particle itself or its payload is an object of analysis performed using the system. In certain embodiments, the payload particle comprises a payload of interest.

The payload of interest may be any product of interest which can be associated with the payload particle. The payload may for example be bound to the outside of the payload particle, entrapped in cavities or pores of the payload particle, or encapsulated within the payload particle. In preferred embodiments, the payload is encapsulated within the payload particle.

In certain embodiments, the payload of interest is a biological cell. The payload may be one or more than one cell. In particular, the payload is exactly one cell or two cells, such as a pair of cells. The cell may be a eukaryotic cell or a prokaryotic cell, preferably a mammalian cell, more preferably a human cell. The cell may be of any cell type. Suitable examples of cell types include cells of the immune system, cells related to different types of cancer, cells of the nervous system, and stem cells. In particular, the cell is a viable cell.

In specific embodiments, the payload particle comprises or - except for the payload - consists of a hydrogel matrix. The material of the payload particle may in particular include a synthetic polymer and/or a natural polymer. Especially, the material is suitable for cell- encapsulation. In certain embodiments, the material of the payload particle comprises a synthetic polymer selected from the group consisting of poly(ethylene glycol) (PEG), poly(ethylene oxide) (PEO), poly(vinyl alcohol) (PVA), polypropylene fumarate) (PPF), poly(acrylic acid) (PAA), poly(acrylic acid-co-acrylamide) (PAAAm), polyacrylamide (PAAm), polylactic acid, polyglycolic acid, and polyoxazoline. In further embodiments, the material of the payload particle comprises a natural polymer selected from the group consisting of agarose, chitosan, collagen, and alginate. In specific embodiments, the material of the payload particle comprises a mixture of at least two different polymers. Suitable polymers and materials are disclosed, for example, in WO 2019/048714 A2. These hydrogel matrices and polymers are especially suitable for encapsulating cells.

In certain embodiments, the matrix of the payload particle has a stiffness represented by Young's moduli (E) in the range of from 300 to 5400 Pa.

The use of payload particles as described herein enables the linkage between functional phenotypes and gene expression analysis in physiological 3D environments. 3D cell culture models gained significant relevance in the last years due to their bio-compatibility, tissue like water content, high porosity, permeability, and in mimicking mechanical properties of the extracellular matrix resulting in a higher physiological relevance. In addition, embedding cells into micro 3D matrices eases cell retrieval after cell cultivation as the hydrogel acts as a uniform vehicle which is insensitive towards cell size thereby making this format compatible with prokaryotes and eukaryotes. In addition, the uniformity of the payload particles has significant advantages for controlling microfluidic flow rates. This enables the usage of the same microfluidic chip for all cell-types.

To link the functional phenotype of a cell to its downstream gene expression profile and genotype, it is crucial that the retrieval process does not alter the expression profile during the isolation process. The payload particle acts as a protective vehicle for transportation of cells as the hydrogel surrounding a cell protects it from shear forces. The small size of the payload particles allows their transport and handling within microfluidic devices. Moreover, the hydrogel is acting as a 3D microenvironment which can give essential stimuli to cultivated cells during the retrieval process (see Mulas, C. etai (2020) Lab on a Chip 20: 2580-2591).

The linkage between the functional phenotype and the underlying genotype of suspension cells, especially cells from the haematopoietic system, is hampered by their floating characteristics making a time-lapse optical analysis and subsequent cell retrieval difficult. Therefore, commercially available systems are not compatible to those cell types limiting the scope of the device to adherent cells. In comparison, cell-laden payload particles as described herein can be efficiently washed by perfusion without affecting and removing encapsulated cells, thereby generating more homogeneous culture conditions and offering a reliable way for the analysis of non-adherent and suspension cells.

The invention overcomes significant technical challenges thereby making microfluidic cell culture procedures accessible for downstream analysis such as next-generation sequencing. By integrating a payload particle consisting of hydrogel polymers and components necessary for the cell-retrieval, the invention overcomes mentioned limitations regarding high production cost and the necessity of extensive peripheral equipment. In comparison to the prior art methods, the components which are crucial for the cell-retrieval are not part of the microfluidic chip but are all incorporated into the retrieval bead polymer. This results in a very cost efficient and fast production of the technology. The payload particles can be generated at high speed and minimum cost resulting in an almost infinite availability of the technology.

Capturing analytes

In certain embodiments, the system further comprises a means for capturing analytes. Analytes in particular are compounds and agents released by the payload of the payload particle. The means for capturing analytes may be part of or associated with the positioning particle. Alternatively, the means for capturing analytes may be part of or associated with a capture particle.

Hence, in certain embodiments the system further comprises a capture particle positioned within the microfluidic channel. In particular, the capture particle is positioned adjacent to or in the vicinity of the payload particle. For example, the capture particle may be located between the payload particle and the positioning particle or the payload particle may be located between the capture particle and the positioning particle. In particular, the capture particle and the payload particle are positioned within the microfluidic channel at a distance of 200 pm or less, preferably 100 pm or less, and more preferably 20 pm or less. Most preferably, the capture particle and the payload particle are in contact with each other. In specific embodiments, the capture particle is moved together with the payload particle.

In specific embodiments, the capture particle is capable of capturing analytes released from the payload of the payload particle. In alternative embodiments, the positioning particle is capable of capturing analytes released from the payload of the payload particle. In other embodiments, the capture particle as well as the positioning particle is capable of capturing analytes released from the payload of the payload particle. In these embodiments, the positioning particle and the capture particle may capture different analytes or the same analytes. In certain embodiments where the system comprises at least two positioning particles, in particular one or more of the positioning particles, especially all of the positioning particles, are capable of capturing analytes released from the payload of the payload particle.

Means for capturing analytes include, for example, capture molecules. These capture molecules may be attached to the positioning particle and/or the capture particle. Alternatively or additionally, the capture molecules may be attached to another structure, such as a smaller particle, which is associated with the positioning particle and/or the capture particle. Said other structure may for example be enclosed within the matrix of the positioning/capture particle. Suitable capture molecules are in particular selected from the group consisting of antibodies, antibody fragments, aptamers, receptor proteins, and ligands. The capture molecules may be attached to the material of the particles, especially to the polymers of the hydrogel matrix of the particles, by covalent bonds or intermolecular interactions. In certain embodiments, the capture molecules are covalently coupled to the polymer matrix of the positioning particle or the capture particle. The positioning particle and/or the capture particle may comprise only one type of capture molecule or a set of different capture molecules.

The analytes to be captured may be any molecules or substances released by the payload. In embodiments where the payload is one or more biological cells, the analytes preferably are selected from the group comprising peptides, polypeptides, proteins, carbohydrates, nucleic acids, small organic molecules and lipids. In particular, the analytes are proteins secreted by the biological cell(s) being the payload of interest. The analytes may be selected from the group consisting of cytokines, growth factors, chemokines, interferons (INF), interleukins (IL), lymphokines, and tumor necrosis factor (TNF). In specific embodiments, the analytes are selected from the group consisting of interleukins (ILs), including I L- 1 a , I L- 1 b , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL- 32, IL-33, IL-34, IL-35, IL-36a, II_-36b, IL-36y, IL-37, IL-1Ra, IL-36Ra and IL-38; interferons (INFs), including type I IFNs (such as IFN-a (further classified into 13 different subtypes such as IFN-a1, -a2, -a4, -a5, -a6, -a7, -a8, -a10, -a13, -a14, -a16, -a17 and -a21), and IFN-b, IFN-d, IFN-e, IFN-z, IFN-k, IFN-V, IFN-T, IFN-w), type II IFN (such as IFN-y) and type III IFNs (such as IFN-A1 and IFN-A2/3,); tumor necrosis factors (TNF), such as TNF-a, TNF-b, CD40 ligand (CD40L), Fas ligand (FasL), TNF-related apoptosis inducing ligand (TRAIL), and LIGHT; chemokines, including CCL1, CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9/CCL10, CCL11, CCL12, CL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, XCL1, XCL2, CX3C, and CX3CL1 ; other cytokines, such perforin, granzyme, MCP-1, MCP-2, MCP-3. Rantes, IP-10, Osteopontin, MIP-1a, MIP-1b, MIP-2, MIP-3a, MIP-5, EGF, VEGF, IGF, G-CSF, GM-CSF, Eotaxin, PDGF, Leptin, and Flt-3; and/or combinations thereof. Particular analytes of interest include EGF, VEGF, CCL2, CCL5, IL-6 and IL-10. For instance, in the beginning of an experiment using the system described herein growth factors such as EGF and VEGF are analyzed, in the middle of the experiment, chemokines such as CCL2 and CCL5 are analyzed, and in the end of the experiment, interleukins such as IL-6 and IL-10 are analyzed.

Suitable analytes and capture molecules and their integration into hydrogel particles are described, for example, in WO 2020/183015 A1. The microfluidic system for analyzing molecules secreted by a payload

The system according to the first aspect in certain embodiments comprises a first capture particle and a second capture particle positioned within the microfluidic channel. One or both of the capture particles may additionally be a positioning particle. In specific embodiments, the positioning particle of the system is the first or the second capture particle. In these embodiments, the system, especially the system for controlling the positioning and movement of a particle for microanalysis, may be a system for analyzing molecules secreted by a payload of interest as described herein.

According to a fifth aspect, the present invention provides a system for analyzing molecules secreted by a payload of interest comprising a microfluidic channel comprising an inlet and an outlet, and positioned within said microfluidic channel a payload particle comprising a payload of interest, a first capture particle and a second capture particle; wherein the capture particles are capable of capturing analytes released from the payload of the payload particle; and wherein the payload particle and the capture particles are positioned within the microfluidic channel in the direction of from inlet to the outlet in the order of

(i) payload particle,

(ii) first capture particle, and

(iii) second capture particle.

The system for analyzing molecules secreted by a payload of interest in particular is a microfluidic system as described herein. The payload particle in particular is a payload particle as described herein. The first capture particle and the second capture particle in particular are capture particles as described herein.

The system for analyzing molecules secreted by a payload of interest comprises a payload particle and two capture particles within the microfluidic channel. The system may comprise more than one payload particle and/or more than two capture particles. The multiple payload particles and multiple capture particles may be present in the same and/or in different microfluidic channels of the system. In preferred embodiments, one payload particle and two capture particles form a group, wherein the first capture particle captures the analytes of the payload of the paired payload particle and the second capture particle captures the analytes present in the system background. In the following, capture particles and payload particle especially refer to the particles of a group of two capture particles and one payload particle.

In certain embodiments, the system for analyzing molecules secreted by a payload of interest comprises a plurality of groups positioned within said microfluidic channel, wherein each group comprising exactly one payload particle and two capture particles. In certain embodiments, the capture particles and the payload particle are adjacent to each other or in the vicinity of each other in the microfluidic channel. In particular, the capture particles and the payload particle are positioned within the microfluidic channel at a distance of 200 pm or less, preferably 100 pm or less, and more preferably 20 pm or less. Most preferably, the capture particles and the payload particle are in contact with each other.

In certain embodiments, the capture particles are fixed at their positions in the microfluidic channel. In particular, the capture particles are wedged in the microfluidic channel due to their size. Especially, the capture particles are not moved by a microfluidic flow applied to the system or the microfluidic channel. In certain embodiments, the payload particle is fixed at its position in the microfluidic channel. In particular, the payload particle is wedged in the microfluidic channel due to its size. In specific embodiments, the capture particles and/or the payload particle are fixed at specific positions in the microfluidic channel. These positions for example have a smaller diameter than other parts of the microfluidic channel or are surrounded by parts of the microfluidic channel with smaller diameters. Due to such designs, a force has to be applied to the capture particles and/or the payload particle in order to move them from their position. In certain embodiments, these specific positions are positions within a microfluidic bead trap. Suitable designs of the microfluidic channel are described, for example in DE 102020004660.6.

In certain embodiments, the first and/or the second capture particle of the system for analyzing molecules secreted by a payload of interest additionally is a positioning particle as described herein. In further embodiments, the system for analyzing molecules secreted by a payload of interest comprises a positioning particle as described herein in addition to the capture particles in the group of particles. Hence, in certain embodiments the system for analyzing molecules secreted by a payload of interest further comprises a positioning particle positioned within the microfluidic channel. In particular, the positioning particle is positioned adjacent to or in the vicinity of the payload particle or the second capture particle. For example, the positioning particle may be located behind the second capture particle in the direction from inlet to outlet of the microfluidic channel. In particular, the positioning particle and the second capture particle are positioned within the microfluidic channel at a distance of 200 pm or less, preferably 100 pm or less, and more preferably 20 pm or less. Most preferably, the positioning particle and the second capture particle are in contact with each other. In specific embodiments, the capture particles are moved together with the payload particle. In preferred embodiments, both the first and the second capture particles additionally are also positioning particles.

In embodiments where the system for analyzing molecules secreted by a payload of interest comprises further groups of one payload particle and two capture particles, in each of these groups at least one, in particular both of the capture particles are additionally also positioning particles. Properties of the particles

The particles of the system, in particular the positioning particle, the payload particle and the optional capture particle, generally may be any type of particles as long as they are capable of exerting the functions described herein. In specific embodiments, the particles of the system are elastic particles. In particular, the particles have a stiffness represented by Young's moduli (E) in the range of from 300 to 5400 Pa.

In certain embodiments, the particles of the system are substantially spherical. In particular, the particles have a diameter in the range of from 1 to 200 pm, preferably from 30 to 150 pm, more preferably from 50 to 100 pm. For example, the particles have a diameter of about 80 pm. In specific embodiments, the particles have a diameter which is similar to the diameter of the microfluidic channel. For example, the diameter of the particles of the system is within +/- 10% of the diameter of the microfluidic channel, especially within +/- 5%. In certain embodiments, the diameter of the particles is chosen so that the particles fit into predefined positions within the microfluidic channel, for example positions in a positioning device. In specific embodiments, the positioning particles have a smaller diameter than the payload particle.

In preferred embodiments, the particles of the system are hydrogel particles. In particular, a hydrogel particle is composed of a hydrogel matrix. The hydrogel matrix may comprise a synthetic polymer or a natural polymer. In certain embodiments, the hydrogel matrix comprises a synthetic polymer selected from the group consisting of poly(ethylene glycol) (PEG), poly(ethylene oxide) (PEO), poly(vinyl alcohol) (PVA), polypropylene fumarate) (PPF), poly(acrylic acid) (PAA), poly(acrylic acid-co-acrylamide) (PAAAm), polyacrylamide (PAAm), polylactic acid, polyglycolic acid, and polyoxazoline. In further embodiments, the hydrogel matrix comprises a natural polymer selected from the group consisting of agarose, chitosan, collagen, and alginate. In specific embodiments, the hydrogel matrix comprises a mixture of at least two different polymers. Suitable hydrogel matrices are disclosed, for example, in WO 2019/048714 A2. In specific embodiments, the hydrogel matrix comprises poly (acrylic acid) polymers and/or agarose.

In specific embodiments, one or more of the particles of the system comprises nanoparticles. In particular, the positioning particle comprises nanoparticles. The nanoparticles may be any nanoparticles known in the art. "Nanoparticles" as used herein refer to particles which have a diameter in the nano- or micrometer range. Especially, the nanoparticles are smaller than the particles of the system. For example, the nanoparticles have a diameter in the range of from 1 nm to 100 pm, preferably from 100 nm to 10 pm, more preferably from 1 pm to 10 nm. The diameter of the nanoparticles in particular refers to their largest diameter. In certain embodiments, the nanoparticles are bound to the particle of the system with an equilibrium dissociation constant of less than 10 ^-12 M. In certain embodiments, the positioning particle comprises only one nanoparticle. In these embodiments, the nanoparticle preferably has a size in the range of 1 pm to 50 pm, especially 5 pm to 20 pm. This one nanoparticle may in particular be a magnetic nanoparticle.

The nanoparticles are in particular used to provide the particles of the system with specific properties. In specific embodiments, the nanoparticles are used for rendering the positioning particle actuatable. For example, magnetic nanoparticles render the positioning particle responsive to a magnetic field. Respective nanoparticles are described herein above concerning the positioning particle. The features of these nanoparticles also apply here. Furthermore, the nanoparticles may be loaded with basic cargo such as NaOH, or with acidic cargo such as HCI or acetic acid. By initiating release of the cargo, the local pH value is altered, resulting for example in swelling or shrinking of the positioning particle or in release of gas.

Furthermore, the nanoparticles may be used for improving identification of the particles, for heating the particles, and/or for plasmonic effects. In certain embodiments, the nanoparticles comprise of gold and/ or silver to use plasmonic principles. In certain embodiments, the nanoparticles comprise a material selected from the group consisting of gold, silver, silica, quantum dots, and FesCU.

The method for moving a payload particle

The system according to the first aspect of the present invention in particular is used for moving the payload particle to or away from a predefined position in the microfluidic channel. Especially, the payload particle or its payload are analyzed and/or manipulated at the predefined position.

In a further aspect, the present invention provides a method for moving a payload particle in a microfluidic channel, comprising the steps of

(i) providing a payload particle and a positioning particle in a microfluidic channel;

(ii) initiating movement of the payload particle by actuating the positioning particle.

The method may in particular be performed using the system as defined herein.

The embodiments, features and examples described herein for the other aspects, especially for the system, also likewise apply to the method for moving a payload particle in a microfluidic channel. In particular, a system according to the first aspect of the invention is used in the method for moving a payload particle in a microfluidic channel.

In certain embodiments, the method further comprises the step of applying a microfluidic flow to the microfluidic channel. This further step may be performed prior to step (i) or between step (i) and step (ii). The microfluidic flow may be applied using a pressure gradient, a micropump or using capillary forces. The microfluidic flow in particular is maintained during step (ii). Applying a microfluidic flow to the microfluidic channel in particular means that a microfluidic flow is generated within microfluidic channels of the system, and that said microfluidic flow would run through the microfluidic channel comprising the payload particle and the positioning particle if the positioning particle does not block the microfluidic flow. The microfluidic flow may be constant throughout the method or may change during the method. In certain embodiments, the strength of the microfluidic flow is controlled. In alternative embodiments, no microfluidic flow is applied to the microfluidic channel during step (ii) of the method or throughout the entire method.

In specific embodiments, the payload particle is moved to or from a position for analyzing and/or manipulating the payload of the payload particle. In certain embodiments, the method further comprises the step of analyzing and/or manipulating the payload of the payload particle. This further step may be performed between steps (i) and (ii) or after step (ii). If it is performed between steps (i) and (ii), the payload particle is moved away from a position for analyzing and/or manipulating the payload of the payload particle in step (ii). If the further step is performed after step (ii), the payload particle is moved to a position for analyzing and/or manipulating the payload of the payload particle in step (ii). Hence, actuating the positioning particle and moving the payload particle may be used to move the payload particle out of a position in which it was analyzed before its movement, or to move the payload particle into a position in which it will be analyzed after its movement.

In specific embodiments, actuating the positioning particle is achieved by using a magnetic field. Especially, actuating the positioning particle in step (ii) includes moving the positioning particle using a magnetic field. In these embodiments, the positioning particle is responsive to a magnetic field. In particular, the payload particle is moved by the movement of the positioning particle. The positioning particle may be moved by moving a magnet relative to the microfluidic channel and/or by turning a magnet on or off.

In certain embodiments, the method includes the step of moving the positioning particle towards the payload particle. Thereby, the payload particle is pushed in the direction of the movement of the positioning particle, either by direct contact to the positioning particle, or by the increased pressure in the fluid between the payload particle and the positioning particle caused by the movement of the positioning particle. In further embodiments, the method includes the step of moving the positioning particle away from the payload particle. Thereby, the payload particle is moved in the direction of the movement of the positioning particle, especially by the undertow created by the movement of the positioning particle, and/or by a microfluidic flow applied to the microfluidic channel.

In specific embodiments, the positioning particle is responsive to a magnetic field and the system used in the method for moving a payload particle in a microfluidic channel is a system according to the first aspect of the invention comprising an optical device to which a source of a magnetic field is attached. Step (ii) in particular includes:

(iia) providing the optical device in a position where the source of the magnetic field is sufficiently remote from the microfluidic channel so that the positioning particle is not actuated by the magnetic field;

(iib) adjusting the position of the microfluidic channel relative to the source of the magnetic field so that the positioning particle is actuated in a desired manner once the source of the magnetic field is brought into range of the microfluidic channel in step (iic); and

(iic) bringing the source of the magnetic field into range of the microfluidic channel so that the magnetic field actuates the positioning particle.

In particular, in step (iib) the microfluidic channel is moved relative to the source of the magnetic field in the X and/or Y axis, wherein the microfluidic channel and optionally the microfluidic chip extends in the X and/or Y axis; and in step (iic) the source of the magnetic field is moved relative to the microfluidic channel in the Z axis. The source of the magnetic field is in particular adjusted to a position in step (iib) to which the positioning particle shall be moved. In step (iic), this movement is effected. By the movement of the positioning particle also movement of the payload particle is initiated. The source of the magnetic field in particular is a magnetizable needle. In step (iib), especially the position of the tip of the magnetizable needle is adjusted so that it is located at the position to which the positioning particle shall move. The optical device in particular is an objective. Adjustment of the position in step (iib) may especially be monitored using the optical device.

In specific embodiments, actuating the positioning particle is achieved by applying light to the positioning particle. In these embodiments, the positioning particle is responsive to light. In particular, actuating the positioning particle in step (ii) includes moving and/or switching on or off of a light source. The light may cause the positioning particle to

(i) shrink, allowing a microfluidic flow applied to the microfluidic channel to pass and/or move the positioning particle, and move the payload particle;

(ii) swell, whereby the payload particle is pushed away from the positioning particle, either by direct contact to the positioning particle, or by the increased pressure in the fluid between the payload particle and the positioning particle caused by the swelling of the positioning particle; or

(iii) release gas, forming a bubble in the microfluidic channel, whereby the payload particle is pushed away by the bubble, either by direct contact or by the increased pressure in the fluid between the payload particle and the bubble. In certain embodiments, step (i) of the method for moving a payload particle in a microfluidic channel comprises

(ia) providing at least two positioning particles in a microfluidic channel;

(ib) crosslinking the at least two positioning particles with each other; and

(ic) introducing a payload particle into the microfluidic channel.

In these embodiments, step (i) comprises providing a payload particle and at least two positioning particles in a microfluidic channel. The method may in particular be performed using the system as defined herein, wherein the system comprises at least two positioning particles within the microfluidic channel, wherein the positioning particles are crosslinked with each other.

In embodiments wherein in step (i) at least two positioning particles are provided, the at least two positioning particles are capable of being crosslinked with each other. However, the at least two positioning particles are not crosslinked with each other in step (ia). The at least two positioning particles are especially positioned in direct vicinity to each other in step (ia) so that crosslinking of the positioning particles via crosslinking agents on their surface is possible in step (ib). In particular, the positioning particles are located at predefined positions within the microfluidic channel, wherein said predefined positions are directly adjacent to each other.

In certain embodiments, the at least two positioning particles comprise crosslinking agents on their surface. These crosslinking agents are capable of forming covalent bonds with the crosslinking agents on the surface of another positioning particle once activated. In specific embodiments, the crosslinking agents do not react to form crosslinks when they are not activated. The crosslinking agents are in particular not activated during step (ia).

In step (ib), the at least two positioning particles are crosslinked with each other. In certain embodiments, crosslinking of the positioning particles is achieved by activating crosslinking agents on their surface. Upon activation, the crosslinking agents of adjacent positioning particles react with each other and form covalent bonds. The crosslinking agents may be activated by applying an activating agent to the microfluidic channel comprising the positioning particles. Thus, in certain embodiments step (ib) comprises applying an activating agent to the positioning particles which initiates a crosslinking reaction between the crosslinking agents of the positioning particles. For example, the activating agent may be added to a liquid which is introduced into the microfluidic channel via a microfluidic flow.

Suitable examples of crosslinking reactions are selected from: (i) a covalent bond formation, preferably selected from (aa) enzymatically catalyzed reactions, such as reactions catalyzed with transglutaminase factor Xllla, (bb) not-enzymatically catalyzed reactions, such as click chemistry or photo-catalyzed reactions and/or (cc) uncatalyzed reactions, such as copper- free highly selective click chemistry, Michael-type addition or Diels-Alder conjugation; (ii) non-covalent bond formation, preferably selected from (aa) hydrogen bonds, preferably formed by nucleic acids or nucleic acid analogs, (bb) hydrophobic interactions, (cc) Van-der- Waals interactions and (dd) electrostatic interactions; and (iii) combinations of the foregoing. Suitable crosslinking agents for the crosslinking reaction are carboxyl, thiol and amine groups, peptides, nucleic acids or unsaturated imides. Suitable activating agents for initiating the crosslinking reaction include acids, alkaline solution, enzymes, reducing agents, oxidizing agents, miscellaneous chemical agents, electromagnetic waves such as UV light, carboxy-, thiol-, amine-, or unsaturated imide-functionalized components, preferably polyethylene glycol (PEG) such as poly(ethylene glycol) bis(amine) or poly(ethylene glycol) dithiol or di(N- succinimidyl) functionalized components or dithiol moieties such as di-thiodipropionic acid di(N-hydroxysuccinimide ester or carboxyfunctionalized disulfides such as 2-carboxyethyl disulfide or hetero-bifunctional reactive components such as 3-(maleimido)-propionic acid N- hydroxysuccinimide ester.

In step (ic), a payload particle is introduced into the microfluidic channel. In certain embodiments, the payload particle is transported into the microfluidic channel via a microfluidic flow applied to the microfluidic channel. Upon introduction into the microfluidic channel, the payload particle is in particular located in the vicinity of the positioning particles. In particular, the positioning particles and the payload particle are positioned within the microfluidic channel at a distance of 200 pm or less, preferably 100 pm or less, and more preferably 20 pm or less. Most preferably, the positioning particles and the payload particle are in contact with each other, wherein especially the payload particle is contacted by one positioning particle and this positioning particle is further crosslinked to the other positioning particle. In certain embodiments, the payload particle is located at a predefined position within the microfluidic channel, wherein said predefined position is directly adjacent to a predefined position of one of the positioning particles.

In step (ii), movement of the payload particle is initiated by actuating the positioning particles. Actuating the positioning particles and initiating movement of the payload particle may be done as described herein.

The method for detecting analytes

The system according to the fifth aspect of the present invention in particular is used for analyzing the payload of interest present in the payload particle. This analysis especially includes capturing analytes which are released by the payload using capture particles. Thus, according to a sixth aspect, the present invention provides a method for detecting analytes released by a payload, comprising the steps of (i) providing a system according to the fifth aspect of the invention;

(ii) incubating the payload of interest in the system for a desired time under conditions at which the payload of interest may release analytes;

(iii) determining the amount of analytes captured by the first capture particle and determining the amount of analytes captured by the second capture particle;

(iv) calculating the amount of analytes released by the payload of interest based on the amount of analytes captured by the first and second capture particles as determined in step (iii), wherein the amount of analytes captured by the second capture particle serves as background signal.

The embodiments, features and examples described herein for the other aspects, especially for the system, also likewise apply to the method for detecting analytes released by a payload.

In certain embodiments, the payload of interest is a biological cell. The payload may be one or more than one cell. In particular, the payload is exactly one cell or two cells, such as a pair of cells. The cell may be a eukaryotic cell or a prokaryotic cell, preferably a mammalian cell, more preferably a human cell. The cell may be of any cell type. Suitable examples of cell types include cells of the immune system, cells related to different types of cancer, cells of the nervous system, and stem cells. In particular, the cell is a viable cell.

In certain embodiments, the system comprises further groups of one payload particle and two capture particles. These further payload particles also comprise one or more cells as payload. In certain embodiments, said cells secrete the same analytes as the cells provided in step (i) of the method. In particular, the capture particles of these further groups are capable of capturing the same analytes as the capture particles provided in step (i) of the method. In specific embodiments, at least one of the further groups is in fluid connection with, in particular within the same microfluidic channel as, the group of the payload particle and the first and second capture particles analyzed in steps (i) to (iv). In particular, at least one of the further groups is within the same microfluidic channel as the group analyzed in steps (i) to (iv).

A microfluidic flow from the inlet to the outlet may be applied to the microfluidic channel during step (ii). In certain embodiments, no or substantially no microfluidic flow is applied to the microfluidic channel during step (ii).

Determining the amount of analytes captured by the capture particles may be performed by any suitable means. Methods for determining the amount of captured analytes are well known to the skilled person. For example, the captured analytes may be bound by a detection agent. Detection agents are capable of binding to the analytes captured by the capture particles and comprise a detectable label. In particular, detection agents specifically bind to a certain analyte, but not to different analytes. Exemplary detection agents include antibodies or antibody fragments, binding partners, ligands and the like, which comprise a label such as a dye, a fluorophore, a radionuclide, or a specific nucleotide sequence. In certain embodiments, step (iii) includes applying a detection agent to the capture particles. One or more different detection agents may be applied, simultaneously or subsequently. Applying the detection agent for example may be performed by adding the detection agent to a microfluidic flow applied to the microfluidic channel. After this, excess unbound detection agent may be washed away using the microfluidic flow without added detection agent. Step (iii) may further include detecting the amount of detection agent bound to the capture particles. This may be performed within the microfluidic channel, or the capture particles may first be removed from the microfluidic channel and detection of the amount may be performed outside of the microfluidic channel.

In certain embodiments, at least one, in particular both of the capture particles are additionally also positioning particles. In embodiments where the system comprises further groups of one payload particle and two capture particles, in each of these groups at least one, in particular both of the capture particles are additionally also positioning particles. In these embodiments, the method for detecting analytes released by a payload may be combined with the method for moving a payload particle in a microfluidic channel. In these combined methods, step (ii) of the method for moving a payload particle, i.e. initiating movement of the payload particle by actuating the positioning particle(s), may be performed between steps (ii) and (iii) or after step (iv) of the method for detecting analytes released by a payload.

Kits comprising the particles In a further aspect, the present invention provides a kit of parts, comprising a payload particle and a positioning particle; wherein the payload particle and the positioning particle are for use in a microfluidic channel; wherein the positioning particle is capable of being actuated; and wherein actuating the positioning particle initiates movement of the payload particle.

The present invention further provides a kit of parts, comprising (i) a positioning particle and material for producing a payload particle; or

(ii) a payload particle and material for producing a positioning particle; or

(iii) material for producing a payload particle and material for producing a positioning particle; wherein the payload particle and the positioning particle are for use in a microfluidic channel; wherein the positioning particle is capable of being actuated; and wherein actuating the positioning particle initiates movement of the payload particle.

In certain embodiments, the kit of parts comprises at least two positioning particles or material for at least two positioning particles, wherein the positioning particles are capable of being crosslinked with each other.

In specific embodiments, the kit further comprises a capture particle or material for producing a capture particle.

In a further aspect, the present invention provides a kit of parts, comprising a payload particle and at least two capture particles; wherein the payload particle and the capture particles are for use in a microfluidic channel; and wherein the capture particles are capable of capturing analytes released from the payload of the payload particle. In specific embodiments, the capture particles are additionally capable of being actuated; and wherein actuating the capture particles initiates movement of the payload particle.

The present invention further provides a kit of parts, comprising

(i) at least two capture particles and material for producing a payload particle; or

(ii) a payload particle and material for producing at least two capture particles; or

(iii) material for producing a payload particle and material for producing at least two capture particles; wherein the payload particle and the capture particles are for use in a microfluidic channel; and wherein the capture particles are capable of capturing analytes released from the payload of the payload particle. In specific embodiments, the capture particles are additionally capable of being actuated; and wherein actuating the capture particles initiates movement of the payload particle.

In specific embodiments, the kit further comprises a positioning particle or material for producing a positioning particle.

The embodiments, features and examples described herein for the other aspects, especially for the system, also likewise apply to the kits of parts.

The material for producing the positioning particle and/or the payload particle and/or the capture particle may be reagents for forming the particles. For example, the material comprises a hydrogel or reagents for forming a hydrogel. Furthermore, the material for producing the positioning particles may comprise suitable nanoparticles. In certain embodiments, the material for producing the positioning particles or the capture particle may comprise suitable means for capturing one or more analytes of interest, as described above.

Uses of the particles

In a further aspect, the present invention provides the use of a positioning particle for initiating movement of a payload particle in a microfluidic channel; wherein the positioning particle is capable of being actuated; and wherein actuating the positioning particle initiates movement of the payload particle.

The embodiments, features and examples described herein for the other aspects, especially for the system, also likewise apply to the use of a positioning particle for initiating movement of a payload particle in a microfluidic channel.

In certain embodiments, the positioning particle is capable of being crosslinked with another positioning particle. In these embodiments, in particular at least two positioning particles are used for initiating movement of a payload particle in a microfluidic channel.

In a further aspect, the present invention provides the use of a capture particle for capturing analytes released from the payload of the payload particle; and optionally initiating movement of a payload particle in a microfluidic channel, wherein the capture particle is capable of being actuated; and wherein actuating the capture particle initiates movement of the payload particle.

The embodiments, features and examples described herein for the other aspects, especially for the system, also likewise apply to the use of a capture particle for capturing analytes.

Definitions

As used in the subject specification, items and claims, the singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise. The terms “include,” “have,” “comprise” and their variants are used synonymously and are to be construed as non-limiting. Further components and steps may be present. Throughout the specification, where compositions are described as comprising components or materials, it is additionally contemplated that the compositions can in embodiments also consist essentially of, or consist of, any combination of the recited components or materials, unless described otherwise. Reference to "the disclosure" and “the invention” and the like includes single or multiple aspects taught herein; and so forth. Aspects taught herein are encompassed by the term "invention". The term “about”, as used herein, is intended to provide flexibility to a specific value or a numerical range endpoint, providing that a given value may be “a little above” or “a little below” the indicated value accounting for variations one might see in the measurements taken among different instruments, samples, and sample preparations. The term usually means within 5%, and preferably within 1% of a given value or range. The term "about" also includes and specifically refers to the exact indicated number or range.

It is preferred to select and combine preferred embodiments described herein and the specific subject-matter arising from a respective combination of preferred embodiments also belongs to the present disclosure.

Specific embodiments

In the following, specific embodiments of the present invention are described.

Embodiment 1. A system comprising a microfluidic channel and positioned within said microfluidic channel a payload particle and a positioning particle; wherein the positioning particle is capable of being actuated; wherein actuating the positioning particle initiates movement of the payload particle.

Embodiment 2. The system according to embodiment 1, wherein the positioning particle and the payload particle are adjacent to each other or in the vicinity of each other in the microfluidic channel.

Embodiment 3. The system according to embodiment 1 or 2, wherein the positioning particle and the payload particle are positioned within the microfluidic channel at a distance of 200 pm or less, preferably 100 pm or less, and more preferably 20 pm or less; most preferably the positioning particle and the payload particle are in contact with each other.

Embodiment 4. The system according to any one of embodiments 1 to 3, wherein the positioning particle in a resting state is fixed at its position in the microfluidic channel.

Embodiment 5. The system according to embodiment 4, wherein the positioning particle in its resting state prevents the payload particle from moving.

Embodiment 6. The system according to embodiment 4 or 5, wherein the positioning particle is wedged in the microfluidic channel due to its size.

Embodiment 7. The system according to any one of embodiments 1 to 6, wherein the payload particle is fixed at its position in the microfluidic channel. Embodiment 8. The system according to embodiment 7, wherein the payload particle is wedged in the microfluidic channel due to its size.

Embodiment 9. The system according to any one of embodiments 1 to 8, wherein the positioning particle is responsive to a magnetic field and wherein actuating the positioning particle includes moving the positioning particle within the microfluidic channel using a magnetic field.

Embodiment 10. The system according to embodiment 9, wherein the payload particle is moved by the movement of the positioning particle.

Embodiment 11. The system according to embodiment 9 or 10, wherein when the positioning particle is moved towards the payload particle, the payload particle is pushed in the direction of the movement of the positioning particle, either by direct contact to the positioning particle, or by the increased pressure in the fluid between the payload particle and the positioning particle caused by the movement of the positioning particle.

Embodiment 12. The system according to any one of embodiments 9 to 11, wherein when the positioning particle is moved away from the payload particle, the payload particle is moved by the undertow created by the movement of the positioning particle, and/or by a microfluidic flow applied to the microfluidic channel.

Embodiment 13. The system according to any one of embodiments 9 to 12, wherein the positioning particle comprises magnetic nanoparticles.

Embodiment 14. The system according to embodiment 13, wherein the magnetic nanoparticles are ferromagnetic, ferrimagnetic, paramagnetic or superparamagnetic or diamagnetic, and/or have a high uniaxial magnetocrystalline anisotropy.

Embodiment 15. The system according to embodiment 13 or 14, wherein the magnetic nanoparticles comprise FesCU, Nd, Ni, Co, NdaFenB, tetracyanoquinodimethane, and/or are coated with polyaniline.

Embodiment 16. The system according to any one of embodiments 9 to 15, wherein the system further comprises a magnet as source of the magnetic field.

Embodiment 17. The system according to embodiment 16, wherein the source of the magnetic field is a permanent magnet or an electromagnet, for example a neodymium magnet.

Embodiment 18. The system according to embodiment 16 or 17, further comprising a magnetizable needle which is magnetized by the source of the magnetic field and which tip is at a distance in the range of from 1 to 2000 pm from the positioning particle, preferably from 20 to 1500 pm, more preferably from 100 to 500 pm.

Embodiment 19. The system according to any one of embodiments 16 to 18, wherein the magnet can be moved relative to the microfluidic channel and/or turned on and off for actuating the positioning particle.

Embodiment 20. The system according to embodiment 19, wherein the magnet is fixed in its position and the microfluidic channel is moved in order to change the position of the positioning particle within the microfluidic channel.

Embodiment 21. The system according to any one of embodiments 1 to 20, wherein the positioning particle is responsive to light and wherein actuating the positioning particle includes applying light to the positioning particle.

Embodiment 22. The system according to embodiment 21, wherein the light causes the positioning particle to shrink, allowing a microfluidic flow applied to the microfluidic channel to pass and/or move the positioning particle, and move the payload particle.

Embodiment 23. The system according to embodiment 21, wherein the light causes the positioning particle to swell, whereby the payload particle is pushed away from the positioning particle, either by direct contact to the positioning particle, or by the increased pressure in the fluid between the payload particle and the positioning particle caused by the swelling of the positioning particle.

Embodiment 24. The system according to embodiment 21, wherein the light causes the positioning particle to release gas, forming a bubble in the microfluidic channel.

Embodiment 25. The system according to embodiment 24, wherein the formed bubble has a diameter in the range of from 1 to 500 pm, preferably from 1 to 90 pm.

Embodiment 26. The system according to embodiment 24 or 25, wherein formation of the bubble pushes the payload particle away from the positioning particle.

Embodiment 27. The system according to any one of embodiments 21 to 26, wherein the material of the positioning particle includes one or more selected from the group consisting of poly(N-isopropylacrylamide), poly(N-isopropylmethacrylamide), poly(acrylic acid-co-acrylamide), polyacrylamide, poly(N,N-diethylacrylamide), poly(N,N- dimethylaminoethyl methacrylate), poly(ethylene glycol), dibenzaldehyde-terminated poly(ethylene glycol), poly(methyl vinyl ether), poly(vinyl alcohol), poly(N-vinylcaprolactam), poly(vinylpyrrolidone), and spiropyran derivates. Embodiment 28. The system according to any one of embodiments 21 to 27, wherein the system further comprises a light source.

Embodiment 29. The system according to embodiment 28, wherein the light source is capable of specifically illuminating the positioning particle.

Embodiment 30. The system according to embodiment 28 or 29, wherein the light source can be moved relative to the microfluidic channel and/or turned on and off for actuating the positioning particle.

Embodiment 31. The system according to any one of embodiments 21 to 30, wherein applying light to the positioning particle causes a local change of the pH value, the temperature, the redox potential, and/or the intermolecular bond formation such as van der Waals, hydrogen bridge, and ionic interactions.

Embodiment 32. The system according to any one of embodiments 21 to 31, wherein the light applied to the positioning particle comprises wavelengths in the range from 1 nm to 10 cm, preferably from 100 nm to 1000 nm, more preferably 365 nm to 900 nm.

Embodiment 33. The system according to any one of embodiments 1 to 32, wherein the payload particle comprises a payload of interest.

Embodiment 34. The system according to embodiment 33, wherein the payload of interest is one or more biological cells, in particular one cell or a cell pair.

Embodiment 35. The system according to embodiment 34, wherein the cell is a eukaryotic cell or a prokaryotic cell, preferably a mammalian cell, more preferably a human cell.

Embodiment 36. The system according to embodiment 32 or 33, wherein the cell is capable of secreting analytes.

Embodiment 37. The system according to any one of embodiments 1 to 36, wherein the material of the payload particle includes a synthetic polymer and/or a natural polymer for cell- encapsulation.

Embodiment 38. The system according to embodiment 37, wherein the synthetic polymer is selected from the group consisting of poly(ethylene glycol) (PEG), poly(ethylene oxide) (PEO), poly(vinyl alcohol) (PVA), polypropylene fumarate) (PPF), poly(acrylic acid) (PAA), poly(acrylic acid-co-acrylamide) (PAAAm), polyacrylamide (PAAm), polylactic acid, polyglycolic acid, and polyoxazoline. Embodiment 39. The system according to embodiment 37, wherein the natural polymer is selected from the group consisting of agarose, chitosan, collagen, and alginate.

Embodiment 40. The system according to any one of embodiments 1 to 39, wherein the matrix of the payload particle has a stiffness represented by Young's moduli (E) in the range of from 300 to 5400 Pa.

Embodiment 41. The system according to any one of embodiments 1 to 40, wherein the system further comprises a capture particle positioned within the microfluidic channel.

Embodiment 42. The system according to embodiment 41, wherein the capture particle is positioned adjacent to or in the vicinity of the payload particle.

Embodiment 43. The system according to embodiment 41 or 42, wherein the capture particle and the payload particle are positioned within the microfluidic channel at a distance of 500 pm or less, preferably 100 pm or less, more preferably 20 pm or less; most preferably the capture particle and the payload particle are in contact with each other.

Embodiment 44. The system according to any one of embodiments 41 to 43, wherein the capture particle is moved together with the payload particle.

Embodiment 45. The system according to any one of embodiments 41 to 44, wherein the capture particle is capable of capturing analytes released from the payload of the payload particle.

Embodiment 46. The system according to any one of embodiments 1 to 40, wherein the positioning particle is capable of capturing analytes released from the payload of the payload particle.

Embodiment 47. The system according to embodiment 45 or 46, wherein the analytes to be captured are selected from the group consisting of cytokines, growth factors such as EGF and VEGF, chemokines such as CCL2 and CCL5, and interleukins such as IL-6 and IL-10.

Embodiment 48. The system according to any one of embodiments 45 to 47, wherein the analytes are captured by capture molecules attached to or associated with the capture particle or positioning particle.

Embodiment 49. The system according to embodiment 48, wherein the capture molecules are selected from the group consisting of antibodies, antibody fragments and aptamers.

Embodiment 50. The system according to any one of embodiments 1 to 49, wherein the positioning particle, the payload particle and/or the capture particle are elastic particles. Embodiment 51. The system according to any one of embodiments 1 to 50, wherein the positioning particle, the payload particle and/or the capture particle are hydrogel particles.

Embodiment 52. The system according to embodiment 51, wherein the hydrogel particles are composed of a hydrogel matrix.

Embodiment 53. The system according to embodiment 52, wherein the hydrogel matrix comprises a synthetic polymer or a natural polymer.

Embodiment 54. The system according to embodiment 53, wherein the synthetic polymer is selected from the group consisting of poly(ethylene glycol) (PEG), poly(ethylene oxide) (PEO), poly(vinyl alcohol) (PVA), polypropylene fumarate) (PPF), poly(acrylic acid) (PAA), poly(acrylic acid-co-acrylamide) (PAAAm), polyacrylamide (PAAm), polylactic acid, polyglycolic acid, and polyoxazoline.

Embodiment 55. The system according to embodiment 53, wherein the natural polymer is selected from the group consisting of agarose, chitosan, collagen, and alginate.

Embodiment 56. The system according to any one of embodiments 52 to 55, wherein the hydrogel matrix has a stiffness represented by Young's moduli (E) in the range of from 300 to 5400 Pa.

Embodiment 57. The system according to embodiment 52, wherein the hydrogel matrix comprises poly (acrylic acid) polymers and/or agarose.

Embodiment 58. The system according to any one of embodiments 1 to 57, wherein the particles are substantially spherical.

Embodiment 59. The system according to any one of embodiments 1 to 58, wherein the particles have a diameter in the range of from 1 to 200 pm, preferably from 30 to 150 pm, more preferably from 50 to 100 pm.

Embodiment 60. The system according to any one of embodiments 1 to 59, wherein the capture particles, the payload particle and/or the positioning particle comprises nanoparticles.

Embodiment 61. The system according to embodiment 60, wherein the nanoparticles have a diameter in the range of from 1 nm to 100 pm, preferably from 100 nm to 10 pm, more preferably from 1 pm to 10 nm.

Embodiment 62. The system according to embodiment 60 or 61, wherein the nanoparticles comprise gold, silver, silica, quantum dots, or FesCU. Embodiment 63. The system according to any one of embodiments 60 to 62, wherein the nanoparticles are loaded with basic cargo such as NaOH, or with acidic cargo such as HCI or acetic acid.

Embodiment 64. The system according to any one of embodiments 60 to 63, wherein the nanoparticles are bound to the positioning particle, the payload particle and/or the capture particle with an equilibrium dissociation constant of less than 10 ¹² M.

Embodiment 65. The system according to any one of embodiments 1 to 64, wherein the system comprises a means for applying a microfluidic flow through the microfluidic channel in the direction from the inlet to the outlet.

Embodiment 66. The system according to any one of embodiments 1 to 65, wherein the positioning particle in its resting state blocks a microfluidic flow through the microfluidic channel and/or a section of the microfluidic channel.

Embodiment 67. The system according to any one of embodiments 1 to 66, wherein the microfluidic channel has a diameter in the range of from 1 to 500 pm, preferably from 30 to 200 pm, more preferably from 50 to 120 pm.

Embodiment 68. The system according to any one of embodiments 1 to 67, wherein the breadth and/or the height of the microfluidic channel and/or the breadth and/or height of a confinement within the microfluidic channel is about as large as the diameter of the payload particle and/or the positioning particle.

Embodiment 69. The system according to any one of embodiments 1 to 68, wherein the microfluidic channel is part of a microfluidic chip.

Embodiment 70. The system according to any one of embodiments 1 to 69, wherein the system comprises a plurality of pairs positioned within said microfluidic channel, wherein each pair comprising exactly one payload particle and one positioning particle.

Embodiment 71. The system according to embodiment 70, wherein a positioning particle of a selected pair is capable of being actuated without actuating positioning particles of the other pairs, wherein actuating said positioning particle initiates movement of the payload particle of said selected pair.

Embodiment 72. The system according to any one of embodiments 1 to 71, wherein the positioning particle is not actuated by a microfluidic flow.

Embodiment 73. The system according to any one of embodiments 1 to 72, comprising at least two positioning particles within said microfluidic channel, wherein the positioning particles are crosslinked with each other. Embodiment 74. The system according to embodiment 73, wherein the at least two positioning particles comprise crosslinking agents on their surface.

Embodiment 75. The system according to embodiment 74, wherein the crosslinking agents are selected from the group consisting of carboxy, thiol and amine groups, peptides, nucleic acids and unsaturated imide moieties.

Embodiment 76. The system according to embodiment 74 or 75, wherein the crosslinking agents on one positioning particle are capable of forming covalent bonds with the crosslinking agents on the surface of another positioning particle once activated.

Embodiment 77. The system according to embodiment 76, wherein the crosslinking agents are activated by applying an activating agent to the microfluidic channel comprising the positioning particles.

Embodiment 78. The system according to embodiment 77, wherein the activating agent is selected from the group consisting of acids, alkaline solutions, enzymes, reducing agents, oxidizing agents, miscellaneous chemical agents, electromagnetic waves such as preferably UV light, carboxy-, thiol-, amine-, or unsaturated imide-functionalized components, preferably polyethylene glycol (PEG) such as poly(ethylene glycol) bis(amine) or poly(ethylene glycol) dithiol or di(N-succinimidyl) functionalized components or dithiol moieties such as di- thiodipropionic acid di(N-hydroxysuccinimide ester or carboxyfunctionalized disulfides such as 2-carboxyethyl disulfide or hetero-bifunctional reactive components such as 3- (maleimido)-propionic acid N-hydroxysuccinimide ester.

Embodiment 79. A system comprising a microfluidic channel comprising an inlet and an outlet, and positioned within said microfluidic channel a payload particle comprising a payload of interest, a first capture particle and a second capture particle; wherein the capture particles are capable of capturing analytes released from the payload of the payload particle; and wherein the payload particle and the capture particles are positioned within the microfluidic channel in the direction of from inlet to the outlet in the order of

(i) payload particle,

(ii) first capture particle, and

(iii) second capture particle.

Embodiment 80. The system according to embodiment 79, wherein the capture particles and the payload particle are adjacent to each other or in the vicinity of each other in the microfluidic channel. Embodiment 81. The system according to embodiment 79 or 80, wherein the first capture particle and the payload particle are positioned within the microfluidic channel at a distance of 200 pm or less, preferably 100 pm or less, and more preferably 20 pm or less; most preferably the first capture particle and the payload particle are in contact with each other.

Embodiment 82. The system according to any one of embodiments 79 to 81, wherein the first capture particle and the second capture particle are positioned within the microfluidic channel at a distance of 200 pm or less, preferably 100 pm or less, and more preferably 20 pm or less; most preferably the first capture particle and the second capture particle are in contact with each other.

Embodiment 83. The system according to any one of embodiments 79 to 82, wherein the capture particles and the payload particle are fixed at their positions in the microfluidic channel.

Embodiment 84. The system according to any one of embodiments 79 to 83, wherein the first and/or the second capture particle additionally is a positioning particle capable of being actuated; wherein actuating the positioning particle initiates movement of the payload particle.

Embodiment 85. The system according to any one of embodiments 79 to 83, further comprising a positioning particle capable of being actuated; wherein actuating the positioning particle initiates movement of the payload particle and optionally also of the capture particles.

Embodiment 86. The system according to any one of embodiments 79 to 85, wherein the microfluidic channel comprises at least one further group of one payload particle and two capture particles.

Embodiment 87. The system according to embodiment 86, wherein the further payload particle(s) also comprises a payload of interest.

Embodiment 88. The system according to embodiment 87, wherein the payload of the further payload particle(s) is of the same type of payload as the payload defined in embodiment 79.

Embodiment 89. The system according to any one of embodiments 86 to 68, wherein a positioning particle of a selected group is capable of being actuated without actuating positioning particles of the other groups, wherein actuating said positioning particle initiates movement of the payload particle of said selected group.

Embodiment 90. The system according to any one of embodiments 79 to 89, having one or more of the features defined in any one of claims 1 to 78. Embodiment 91. A method for moving a payload particle in a microfluidic channel, comprising the steps of

(i) providing a payload particle and a positioning particle in a microfluidic channel;

(ii) initiating movement of the payload particle by actuating the positioning particle.

Embodiment 92. The method according to embodiment 91, having one or more of the features of the system as defined in embodiments 1 to 90.

Embodiment 93. The method according to embodiment 91 or 92, further comprising the step of applying a microfluidic flow to the microfluidic channel.

Embodiment 94. The method according to embodiment 93, wherein the microfluidic flow is applied to the microfluidic channel during steps (i) and (ii).

Embodiment 95. The method according to embodiment 93 or 94, wherein a constant microfluidic flow is applied to the microfluidic channel.

Embodiment 96. The method according to any one of embodiments 91 to 95, wherein the payload particle is moved to or from a position for analyzing and/or manipulating the payload of the payload particle.

Embodiment 97. The method according to any one of embodiments 91 to 96, wherein the positioning particle is responsive to a magnetic field, and wherein actuating the positioning particle in step (ii) includes moving the positioning particle using a magnetic field.

Embodiment 98. The method according to embodiment 97, wherein the payload particle is moved by the movement of the positioning particle.

Embodiment 99. The method according to embodiment 97 or 98, including the step of moving the positioning particle towards the payload particle, thereby pushing the payload particle in the direction of the movement of the positioning particle, either by direct contact to the positioning particle, or by the increased pressure in the fluid between the payload particle and the positioning particle caused by the movement of the positioning particle.

Embodiment 100. The method according to embodiment 97 or 98, including the step of moving the positioning particle away from the payload particle, thereby moving the payload particle by the undertow created by the movement of the positioning particle, and/or by a microfluidic flow applied to the microfluidic channel.

Embodiment 101. The method according to any one of embodiments 97 to 100, wherein the positioning particle is moved by moving a magnet relative to the microfluidic channel and/or turning a magnet on or off, wherein the magnet is the source of the magnetic field. Embodiment 102. The method according to any one of embodiments 91 to 96, wherein the positioning particle is responsive to light and wherein actuating the positioning particle includes applying light to the positioning particle.

Embodiment 103. The method according to embodiment 102, wherein the light causes the positioning particle to

(i) shrink, allowing a microfluidic flow applied to the microfluidic channel to pass and/or move the positioning particle, and move the payload particle;

(ii) swell, whereby the payload particle is pushed away from the positioning particle, either by direct contact to the positioning particle, or by the increased pressure in the fluid between the payload particle and the positioning particle caused by the swelling of the positioning particle; or

(iii) release gas, forming a bubble in the microfluidic channel, whereby the payload particle is pushed away by the bubble, either by direct contact or by the increased pressure in the fluid between the payload particle and the bubble.

Embodiment 104. The method according to embodiment 102 or 103, wherein actuating the positioning particle in step (ii) includes moving and/or switching on or off of a light source.

Embodiment 105. The method according to any one of embodiments 91 to 104, wherein in step (i) a payload particle and at least two positioning particles are provided in the microfluidic channel.

Embodiment 106. The method according to embodiment 105, wherein step (i) comprises

(ia) providing at least two positioning particles in a microfluidic channel;

(ib) crosslinking the at least two positioning particles with each other; and

(ic) introducing a payload particle into the microfluidic channel.

Embodiment 107. The method according to embodiment 106, wherein in step (ia) the positioning particles are located at predefined positions within the microfluidic channel, wherein said predefined positions are directly adjacent to each other.

Embodiment 108. The method according to any one of embodiments 105 to 107, wherein the at least two positioning particles comprise crosslinking agents on their surface.

Embodiment 109. The method according to embodiment 108, wherein the crosslinking agents are selected from the group consisting of carboxy, thiol and amine groups, peptides, nucleic acids and unsaturated imide moieties. Embodiment 110. The method according to embodiment 108 or 109, wherein the crosslinking agents on one positioning particle are capable of forming covalent bonds with the crosslinking agents on the surface of another positioning particle once activated.

Embodiment 111. The method according to any one of embodiments 108 to 110, wherein in step (ii) the crosslinking agents on the positioning particles are activated, resulting in crosslinking of the positioning particles.

Embodiment 112. The method according to embodiment 111, wherein the crosslinking agents are activated by applying an activating agent to the microfluidic channel comprising the positioning particles.

Embodiment 113. The method according to embodiment 112, wherein the activating agent is selected from the group consisting of acids, alkaline solutions, enzymes, reducing agents, oxidizing agents, miscellaneous chemical agents, electromagnetic waves such as preferably UV light, carboxy-, thiol-, amine-, or unsaturated imide-functionalized components, preferably polyethylene glycol (PEG) such as poly(ethylene glycol) bis(amine) or poly(ethylene glycol) dithiol or di(N-succinimidyl) functionalized components or dithiol moieties such as di-thiodipropionic acid di(N-hydroxysuccinimide ester or carboxyfunctionalized disulfides such as 2-carboxyethyl disulfide or hetero-bifunctional reactive components such as 3-(maleimido)-propionic acid N-hydroxysuccinimide ester.

Embodiment 114. The method according to any one of embodiments 106 to 113, wherein step (ib) comprises applying an activating agent to the positioning particles which initiates a crosslinking reaction between the crosslinking agents of the positioning particles.

Embodiment 115. The method according to embodiment 114, wherein applying the activating agent comprises adding the activating agent to a liquid which is introduced into the microfluidic channel via a microfluidic flow.

Embodiment 116. The method according to any one of embodiments 106 to 115, wherein in step (ic) the payload particle is transported into the microfluidic channel via a microfluidic flow applied to the microfluidic channel.

Embodiment 117. The method according to any one of embodiments 106 to 116, wherein after step (ic) the payload particle is located in the vicinity of the positioning particles.

Embodiment 118. The method according to embodiment 117, wherein the positioning particles and the payload particle are positioned within the microfluidic channel at a distance of 200 pm or less, preferably 100 pm or less, and more preferably 20 pm or less.

Embodiment 119. The method according to embodiment 118, wherein at least one of the positioning particles and the payload particle are in contact with each other. Embodiment 120. The method according to embodiment 119, wherein the payload particle is located at a predefined position within the microfluidic channel, wherein said predefined position is directly adjacent to a predefined position of one of the positioning particles.

Embodiment 121. A method for detecting analytes released by a payload of interest, comprising the steps of

(i) providing a system according to any one of embodiments 79 to 90;

(ii) incubating the payload of interest in the system for a desired time under conditions at which the payload of interest may release analytes;

(iii) determining the amount of analytes captured by the first capture particle and determining the amount of analytes captured by the second capture particle;

(iv) calculating the amount of analytes released by the payload of interest based on the amount of analytes captured by the first and second capture particles as determined in step (iii), wherein the amount of analytes captured by the second capture particle serves as background signal.

Embodiment 122. The method according to embodiment 121, wherein the payload of interest is one or more biological cells, especially one cell or a cell pair.

Embodiment 123. The method according to embodiment 122, wherein the cell is a eukaryotic cell or a prokaryotic cell, preferably a mammalian cell, more preferably a human cell.

Embodiment 124. The method according to embodiment 122 or 123, wherein the cell is capable of secreting analytes.

Embodiment 125. The method according to any one of embodiments 121 to 124, wherein the analytes are selected from the group consisting of cytokines, growth factors such as EGF and VEGF, chemokines such as CCL2 and CCL5, and interleukins such as IL-6 and IL-10.

Embodiment 126. The method according to any one of embodiments 121 to 125, wherein capture molecules capable of capturing the analytes are attached to or associated with the capture particles.

Embodiment 127. The method according to embodiment 126, wherein the capture molecules are selected from the group consisting of antibodies, antibody fragments and aptamers. Embodiment 128. The method according to any one of embodiments 121 to 127, wherein in step (ii) the payload of interest is incubated for a time sufficient for the analytes released by the payload of interest to reach the first capture particle.

Embodiment 129. The method according to any one of embodiments 121 to 128, wherein in step (ii) the analytes are captured by capture molecules attached to or associated with the first capture particle.

Embodiment 130. The method according to any one of embodiments 121 to 129, wherein step (iii) includes contacting the analytes captured by the capture particles with a detection agent.

Embodiment 131. The method according to embodiment 130, wherein the detection agent is capable of specifically binding to the analytes captured by the capture particles and comprise a detectable label.

Embodiment 132. The method according to embodiment 130 or 131, wherein the detection agent is selected from the group consisting of antibodies, antibody fragments, binding partners, and ligands.

Embodiment 133. The method according to embodiment 131 or 132, wherein the detectable label is selected from the group consisting of dyes, fluorophores, radionuclides, and specific nucleotide sequences.

Embodiment 134. The method according to any one of embodiments 130 to 133, wherein step (iii) includes adding the detection agent to a microfluidic flow applied to the microfluidic channel.

Embodiment 135. The method according to embodiment 134, wherein the method further includes the step of washing the particles after adding the detection agent.

Embodiment 136. The method according to any one of embodiments 130 to 135, wherein step (iii) includes detecting the amount of detection agent bound to the capture particles.

Embodiment 137. The method according to any one of embodiments 121 to 136, wherein the system comprises further groups of one payload particle and two capture particles, wherein the further payload particle also comprises one or more cells as payload which may release the same analytes.

Embodiment 138. The method according to embodiment 137, wherein at least one of the further groups is in fluid connection with the group of the payload particle and the first and second capture particles analyzed in steps (i) to (iv). Embodiment 139. The method according to embodiment 137 or 138, wherein at least one of the further groups is within the same microfluidic channel as the group of the payload particle and the first and second capture particles analyzed in steps (i) to (iv).

Embodiment 140. The method according to any one of embodiments 121 to 139, wherein no or substantially no microfluidic flow is applied to the microfluidic channel during step (ii).

Embodiment 141. A kit of parts, comprising

(i) a payload particle or material for producing a payload particle; and

(ii) a positioning particle or material for producing a positioning particle; wherein the payload particle and the positioning particle are for use in a microfluidic channel; wherein the positioning particle is capable of being actuated; and wherein actuating the positioning particle initiates movement of the payload particle.

Embodiment 142. The kit of parts according to embodiment 141, further comprising a capture particle or material for producing a capture particle.

Embodiment 143. The kit of parts according to embodiment 141 or 142, comprising at least two positioning particles or material for producing at least two positioning particles, wherein the positioning particles are capable of being crosslinked with each other.

Embodiment 144. The kit of parts according to any one of embodiments 141 to 143, wherein the payload particle, the positioning particle and/or the capture particle have one or more of the features as defined in embodiments 1 to 90.

Embodiment 145. A kit of parts, comprising

(i) a payload particle or material for producing a payload particle; and

(ii) at least two capture particles or material for producing at least two capture particles; wherein the payload particle and the capture particles are for use in a microfluidic channel; and wherein the capture particles are capable of capturing analytes released from the payload of the payload particle.

Embodiment 146. The kit of parts according to embodiment 145, wherein the capture particles are additionally capable of being actuated; and wherein actuating the capture particles initiates movement of the payload particle.

Embodiment 147. The kit of parts according to embodiment 145, further comprising a positioning particle or material for producing a positioning particle. Embodiment 148. The kit of parts according to any one of embodiment 145 to 147, wherein the payload particle, the positioning particle and/or the capture particle have one or more of the features as defined in embodiments 1 to 90.

Embodiment 149. Use of a positioning particle for initiating movement of a payload particle in a microfluidic channel; wherein the positioning particle is capable of being actuated; and wherein actuating the positioning particle initiates movement of the payload particle.

Embodiment 150. The use according to embodiment 149, having one or more of the features of the system as defined in embodiments 1 to 90.

Embodiment 151. Use of a capture particle for capturing analytes released from the payload of the payload particle; and optionally for initiating movement of the payload particle in a microfluidic channel; wherein the capture particle is capable of being actuated; and wherein actuating the capture particle initiates movement of the payload particle.

Embodiment 152. The use according to embodiment 151, having one or more of the features of the system as defined in embodiments 1 to 90.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows a section of a microfluidic channel (1) comprising a payload particle (2) with a payload (3) and a magnetically responsive positioning particle (4) adjacent to each other. The particles are located at predefined positions (A and B) within a microfluidic positioning device (11) of the microfluidic channel (1). Upon movement of a magnetic source (5), the magnetically responsive positioning particle (4) is actuated and moved into the direction of the payload particle (2), thereby pushing the payload particle (2) out of its position (B) of the positioning device (11).

Figure 2 shows a section of a microfluidic channel (1) comprising a payload particle (2) with a payload (3) and a magnetically responsive positioning particle (4) adjacent to each other. The particles are located at predefined positions (A and B) within a microfluidic positioning device (11) of the microfluidic channel (1). Upon movement of a magnetic source (5), the magnetically responsive positioning particle (4) is actuated and moved away from the payload particle (2), thereby pulling the payload particle (2) - by the undertow created by the movement of the positioning particle (4) - out of its position (A) of the positioning device (11).

Figure 3 shows a section of a microfluidic channel (1) comprising a payload particle (2) with a payload (3) and a light-responsive positioning particle (4) adjacent to each other. The particles are located at predefined positions (A and B) within a microfluidic positioning device (11) of the microfluidic channel (1). In its non-actuated state, the positioning particle (4) blocks the path of the microfluidic flow (8) in the microfluidic channel (1) so that it cannot reach the payload particle (2) (upper illustration). Upon irradiation of the light-responsive positioning particle (4) with light using a light source (7), the positioning particle (4) is actuated and shrunk, thereby allowing the microfluidic flow (8) in the microfluidic channel (1) to catch the payload particle (2) and push it out of its position (B) of the positioning device (11) (lower illustration).

Figure 4 shows a section of a microfluidic channel (1) comprising a payload particle (2) with a payload (3) and a light-responsive positioning particle (4) adjacent to each other. The particles are located at predefined positions (A and B) within a microfluidic positioning device (11) of the microfluidic channel (1). In its non-actuated state, the positioning particle (4) blocks the path of the payload particle (2), which therefore cannot be pushed by the microfluidic flow (8) in the microfluidic channel (1) (upper illustration). Upon irradiation of the light-responsive positioning particle (4) with light using a light source (7), the positioning particle (4) is actuated and shrunk, thereby no longer blocking the microfluidic channel (1) and allowing the microfluidic flow (8) to push the payload particle (2) out of its position (A) of the positioning device (11) (lower illustration).

Figure 5 shows a section of a microfluidic channel (1) comprising a payload particle (2) with a payload (3) and a light-responsive positioning particle (4) adjacent to each other. The particles are located at predefined positions (A and B) within a microfluidic positioning device (11) of the microfluidic channel (1). Upon irradiation of the light-responsive positioning particle (4) with light using a light source (7), the positioning particle (4) is actuated and swelled, thereby pushing the payload particle (2) out of its position (B) of the positioning device (11) (lower illustration).

Figure 6 shows a section of a microfluidic channel (1) comprising a payload particle (2) with a payload (3) and a light-responsive positioning particle (4) adjacent to each other. The particles are located at predefined positions (A and B) within a microfluidic positioning device (11) of the microfluidic channel (1). Upon irradiation of the light-responsive positioning particle (4) with light using a light source (7), the positioning particle (4) is actuated and releases a gas bubble (9), which pushes the payload particle (2) out of its position (B) of the positioning device (11) (lower illustration).

Figure 7 shows a section of a microfluidic channel (1) comprising a payload particle (2) with a payload (3) and a light-responsive positioning particle (4) adjacent to each other. The particles are located at predefined positions (A and B) within a microfluidic positioning device (11) of the microfluidic channel (1). In its non-actuated state, the positioning particle (4) blocks the path of the payload particle (2), which therefore cannot be pushed by the microfluidic flow (8) in the microfluidic channel (1) (upper illustration). Upon irradiation of the light-responsive positioning particle (4) with light using a light source (7), the positioning particle (4) is actuated and releases a gas bubble (9), which pushes the positioning particle (4) out of its position (B) of the positioning device (11). Thereby, it no longer blocks the microfluidic channel (1) and allows the microfluidic flow (8) to push the payload particle (2) out of its position (A) of the positioning device (11) (lower illustration).

Figure 8 shows a section of a microfluidic channel (1) comprising a payload particle (2) with a payload (3), a magnetically responsive positioning particle (4) and a capture particle (10) adjacent to each other. The particles are located at predefined positions (A, B and C) within a microfluidic positioning device (11) of the microfluidic channel (1). Upon movement of a magnetic source (5), the magnetically responsive positioning particle (4) is actuated and moved into the direction of the payload particle (2), thereby pushing the payload particle (2) and the capture particle (10) out of their positions (B and C) of the positioning device (11).

Figure 9 shows a section of a microfluidic channel (1) comprising a payload particle (2) with a payload (3), a magnetically responsive positioning particle (4) and a capture particle (10) adjacent to each other. The particles are located at predefined positions (A, B and C) within a microfluidic positioning device (11) of the microfluidic channel (1). Upon movement of a magnetic source (5), the magnetically responsive positioning particle (4) is actuated and moved away from the payload particle (2), thereby pulling the payload particle (2) and the capture particle (10) - by the undertow created by the movement of the positioning particle (4) - out of their positions (A and B) of the positioning device (11).

Figure 10 shows a section of a microfluidic channel (1) comprising a payload particle (2) with a payload (3), a light-responsive positioning particle (4) and a capture particle (10) adjacent to each other. The particles are located at predefined positions (A, B and C) within a microfluidic positioning device (11) of the microfluidic channel (1). In its non-actuated state, the positioning particle (4) blocks the path of the microfluidic flow (8) in the microfluidic channel (1) so that it cannot reach the payload particle (2) and the capture particle (10) (upper illustration). Upon irradiation of the light-responsive positioning particle (4) with light using a light source (7), the positioning particle (4) is actuated and shrunk, thereby allowing the microfluidic flow (8) in the microfluidic channel (1) to catch the payload particle (2) and the capture particle (10) and push them out of their position (B and C) of the positioning device (11) (lower illustration).

Figure 11 shows a section of a microfluidic channel (1) comprising a payload particle (2) with a payload (3), a light-responsive positioning particle (4) and a capture particle (10) adjacent to each other. The particles are located at predefined positions (A, B and C) within a microfluidic positioning device (11) of the microfluidic channel (1). In its non-actuated state, the positioning particle (4) blocks the path of the payload particle (2) and the capture particle (10), which therefore cannot be pushed by the microfluidic flow (8) in the microfluidic channel (1) (upper illustration). Upon irradiation of the light-responsive positioning particle (4) with light using a light source (7), the positioning particle (4) is actuated and shrunk, thereby no longer blocking the microfluidic channel (1) and allowing the microfluidic flow (8) to push the payload particle (2) and the capture particle (10) out of their positions (A and B) of the positioning device (11) (lower illustration).

Figure 12 shows a section of a microfluidic channel (1) comprising a payload particle (2) with a payload (3), a light-responsive positioning particle (4) and a capture particle (10) adjacent to each other. The particles are located at predefined positions (A, B and C) within a microfluidic positioning device (11) of the microfluidic channel (1). Upon irradiation of the light-responsive positioning particle (4) with light using a light source (7), the positioning particle (4) is actuated and swelled, thereby pushing the payload particle (2) and the capture particle (10) out of their positions (B and C) of the positioning device (11) (lower illustration).

Figure 13 shows a section of a microfluidic channel (1) comprising a payload particle (2) with a payload (3), a light-responsive positioning particle (4) and a capture particle (10) adjacent to each other. The particles are located at predefined positions (A, B and C) within a microfluidic positioning device (11) of the microfluidic channel (1). Upon irradiation of the light-responsive positioning particle (4) with light using a light source (7), the positioning particle (4) is actuated and releases a gas bubble (9), which pushes the payload particle (2) and the capture particle (10) out of their positions (B and C) of the positioning device (11) (lower illustration).

Figure 14 shows a section of a microfluidic channel (1) comprising a payload particle (2) with a payload (3), a light-responsive positioning particle (4) and a capture particle (10) adjacent to each other. The particles are located at predefined positions (A, B and C) within a microfluidic positioning device (11) of the microfluidic channel (1). In its non-actuated state, the positioning particle (4) blocks the path of the payload particle (2) and the capture particle (10), which therefore cannot be pushed by the microfluidic flow (8) in the microfluidic channel (1) (upper illustration). Upon irradiation of the light-responsive positioning particle (4) with light using a light source (7), the positioning particle (4) is actuated and releases a gas bubble (9), which pushes the positioning particle (4) out of its position (C) of the positioning device (11). Thereby, it no longer blocks the microfluidic channel (1) and allows the microfluidic flow (8) to push the payload particle (2) and the capture particle (10) out of their positions (A and B) of the positioning device (11) (lower illustration).

Figure 15 shows a positioning device (21) comprising a first receptacle (23) and a second receptacle (24) for positioning of a particle. The first receptacle (23) is defined by a first opening (25) and first and second confining portions (29, 30) and the second receptacle (24) is defined by a second opening (31) and second and third confining portions (30, 32). The positioning device comprises a branching point (28) where a particle can either enter the first receptacle (23) or bypass the receptacles through bypass channels (26, 27). Figure 16 shows a positioning device (21) comprising two particles. A positioning particle (40) is located in the second receptacle and a payload particle (41) is located in the first receptacle.

Figure 17 shows a system for single cell analysis. On a microfluidic chip, pairs of a magnetically responsive positioning particle (4) and a payload particle (2) comprising a single cell are captured in positioning device (11) within a microfluidic channel (1). The chip is mounted in a cartridge holder (51) in a horizontal orientation and an objective (50) comprising a magnetic needle (5) is positioned below the chip to monitor the particles in the microfluidic channel. A: The magnetic needle (5) is in a bottom position where the magnetic field is not strong enough to move the positioning particle (4). In this arrangement, the needle tip is moved to the desired spot below the microfluidic channel (1). B: The magnetic needle (5), once at the desired spot below the microfluidic channel (1), is moved upwards so that the magnetic field can attract the positioning particle (4). C: The positioning particle (4) - due to the magnetic attraction - moves to the magnetic needle (5), thereby pushing the payload particle (2) out of its confinement of the positioning device (11). D: The payload particle (2) is washed out of the positioning device (11) by the microfluidic flow applied to the microfluidic channel (1).

Figure 18 shows a section of a microfluidic channel (101) comprising a payload particle (113) and two actuatable positioning particles (114 and 115) adjacent to each other. The particles are located at predefined positions within a microfluidic bead trap (102) of the microfluidic channel (101).

Figure 19 shows a section of a microfluidic channel (1) comprising two positioning devices (11). Each positioning device comprises a payload particle (2) with a payload (3) and first and second capture particles (10) adjacent to each other. The particles are located at predefined positions within the microfluidic bead trap (11) of the microfluidic channel (1). The first capture particle captures the analytes secreted from the payload of the adjacent payload particle. The second capture particle captures the analytes which are generally present in the microfluidic flow (8), e.g. originating from another payload upstream in the microfluidic channel. Thereby, the second capture particle serves as background control which is used to correct the amount of analytes captured by the first capture particle in order to determine the analytes secreted by the respective payload. Reference signs

1 microfluidic channel 29 first confining portion

2 payload particle 30 second confining portion

3 payload 31 second opening

4 positioning particle 32 third confining portion

5 source of a magnetic field 40/41 particle

6 magnetic force 50 objective

7 light source 51 cartridge holder with microfluidic chip

8 microfluidic flow 101 microfluidic channel

9 gas bubble 102 microfluidic positioning device

10 capture particle 108 microfluidic flow 11 microfluidic positioning device 109 first confining portion 21 positioning device 110 second confining portion 22 confinement structure 111 third confining portion

23 the first receptacle 112 fourth confining portion

24 second receptacle 113 payload particle

25 device (first) opening 114 first positioning particle

26/27 bypass channel 115 second positioning particle

28 branch point

EXAMPLES

It should be understood that the following examples are for illustrative purpose only and are not to be construed as limiting this invention in any manner. Example 1 : Formation of particle-laden hydrogel beads

Polyacrylamide (PAAm) hydrogel particles were synthesized using droplet-based microfluidics. An aqueous liquid consisting of a monomer solution and particles of different sizes were dispersed into a continuous phase of HFE-7500 containing 0.4 %(w/v) surfactant. TEMED 0.4 % (v/v) and APS 0.3 % (w/v) were used to initiated hydrogel formation. Droplet formation was performed in a microfluidic flow-focusing device with a channel width of 80 pm. The water-in-oil emulsion was generated by applying a pressure of 150 - 250 mbar to the continuous phase, 150 - 250 mbar to the aqueous phase and 0 - 100 mbar to the outlet. The pressure was generated and controlled by the evorion®CellCity System. After droplet formation, 200pL mineral oil was added on top of the droplet phase, and droplets were allowed to polymerize over night at 65°C by a free radical polymerization reaction. The resulting hydro-gel beads were demulsified by removing both oil phases and adding 400 pL of sterile filtered PBS and 100mI_ PFO to the particle solution. The aqueous phase was filtered by a 100 pm mesh filter (Sysmex, Kobe, Japan).

Example 2: Retrieval of cell-laden particles using light-induced shrinkage or magnetic force

For co-localization of cell-laden hydrogel beads and positioning particles inside the evorion®CellCity BeadPairing Chip, cell-laden agarose beads as well as positioning particles were mixed in PBS with a 1:1 ratio. Each inlet of the BeadPairing chip was filled with 150 pl_ of the prepared hydrogel/particle mixture. Subsequently, the evorion®CellCity Incubator was closed, and trapping was performed by applying a pre-defined pressure profile to all inlet reservoirs. By applying the pressure to the inlets, a flow is generated in each channel of the CellCity Bead PairingChip, which results in the immobilization of the hydrogel beads by a hydrodynamic trapping mechanism within trapping positions. After trapping, channels were washed twice with PBS and filled with cell culture medium. To remove specific cell-laden payload particles, two procedures were tested.

For particle- retrieval by light the equatorial plane of the positioning particle was focused in the field of view. Afterwards the positioning particle was illuminated for two seconds with a laser. By using a laser intensity of 10 mW, a spot size of 3 pm and a wavelength of 561 nm, a shrinkage-effect was induced in the positioning particle. By applying a microfluidic flow, the cell-laden payload particle was pushed out of the trapping position.

For particle- retrieval by magnetic force, a magnetic needle connected to the objective was placed in proximity downstream in the microfluidic channel. Because of the attraction of the positioning particle by the magnetic needle the positioning particle pushed the cell-laden payload particle out of the trapping position (see Figure 17).

Example 3: Crosslinking of positioning particles

For co-localization of cell-laden hydrogel beads and positioning particles inside the evorion®CellCity BeadPairing Chip, cell-laden agarose beads as well as positioning particles were mixed in PBS with a 1:1 ratio. Each inlet of the BeadPairing chip was filled with 150 pl_ of the prepared hydrogel/particle mixture. Subsequently, the evorion®CellCity Incubator was closed, and trapping was performed by applying a pre-defined pressure profile to all inlet reservoirs. By applying the pressure to the inlets, a flow is generated in each channel of the CellCity Bead PairingChip, which results in the immobilization of the hydrogel beads by a hydrodynamic trapping mechanism within trapping positions. After trapping, channels were washed twice with PBS and a second batch of positioning particles is added to the CellCity Bead PairingChip for a second round of immobilization of the hydrogel beads by a hydrodynamic trapping mechanism within trapping positions. After trapping, channels were again washed twice with PBS to remove untapped particles.

For crosslinking of two paired positioning particles a crosslinking initiating agent is applied to the CellCity Bead PairingChip. Alternatively, the chip is illuminated with UV light. After the crosslinking reaction the chip is washed twice with PBS to remove residual crosslinking initiating agent.

Example 4: Determination of analyte secretion of single cells

Secretion of analytes from a single cell was determined using a payload particle comprising a single cell and two capture particles positioned in a bead trap as described, for example, in patent application DE 102022 101 699.4, and as illustrated in Figure 19.

Payload particles are incubated resulting in secretion of analytes by cell(s) located within the payload particles. The volume flow is stopped / significantly reduced within the microfluidic channel so that analyte movement is mainly / only due to diffusion and not perfusion. Analytes bind to a first antibody which is immobilized within capture particle. Since the first capture particle is directly adjacent to the payload particle, the analytes of the cell(s) first get into contact with and are captured by the first antibodies of the first capture particle.

Analytes from a downstream position may get into contact with the second capture particle due to a diffusion to the upstream position.

During incubation, a fluorescently labelled second antibody is provided within an aqueous phase located within the microfluidic channel. As soon as analytes get into contact with a capture particle, the fluorescently labelled second antibody binds to another epitope on the analytes. This results in an accumulation of the fluorescently labelled second antibody within the capture particle, thereby generating a fluorescent signal. Determination of the captured analytes is also described in detail in WO 2020/183015 A1.

The fluorescent signal is inhomogenously distributed among the capture particles due to the diffusion of the analytes. This allows the determination of the source of the secreted analytes and additionally serves as a negative control.