EP4122599A1

EP4122599A1 - System for controlling microparticle movement

Info

Publication number: EP4122599A1
Application number: EP21186702.3A
Authority: EP
Inventors: Robert WEINGARTEN; Sebastian BÜHREN; Hans KLEINE-BRÜGGENEY
Original assignee: Evorion Biotechnologies GmbH
Current assignee: Evorion Biotechnologies GmbH
Priority date: 2021-07-20
Filing date: 2021-07-20
Publication date: 2023-01-25

Abstract

The present invention provides a system for moving a particle of interest in a microfluidic channel, using a second, actuatable particle. The second particle may be actuated using e.g. a magnetic field or light, resulting in an actuated state of the second particle, whereby movement of the particle of interest is initiated.

Description

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 microenvironments 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 Gómez-Sjöberg, R. et al. (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 Gómez-Sjöberg, 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 addition, one significant disadvantage of the prior art methods is the necessity of valve 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 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.
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.

The microfluidic system

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 µm, preferably from 30 to 200 µm, more preferably from 50 to 120 µm. Specifically, the microfluidic channel(s) may have a diameter of about 70 to 100 µm. 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 µm and a height of about 80 µm. 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.
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.
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 µm or less, preferably 100 µm or less, and more preferably 20 µm 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 bead trap. Suitable designs of the microfluidic channel are described, for example in DE 10 2020 004 660.6 .
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

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.

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 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 Fe₃O₄, Nd, Ni, Co, Nd₂Fe₁₄B, 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 µm from the positioning particle, preferably from 20 to 1500 µm, more preferably from 100 to 500 µm. 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, either 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. 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.

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.
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 µm, preferably from 1 to 90 µm.
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 µm, preferably 1 to 10 µm, such as about 3 µm.
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), poly(propylene 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. et al. (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 µm or less, preferably 100 µm or less, and more preferably 20 µm 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.
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 IL-1α, IL-1β, 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-36α, IL-36β, IL-36γ, IL-37, IL-1Ra, IL-36Ra and IL-38; interferons (INFs), including type I IFNs (such as IFN-α (further classified into 13 different subtypes such as IFN-α1, -α2, -α4, -α5, -α6, -α7, -α8, -α10, -α13, -α14, -α16, -α17 and -α21), and IFN-β, IFN-δ, IFN-ε, IFN-ζ, IFN-κ, IFN-ν, IFN-τ, IFN-ω), type II IFN (such as IFN-γ) and type III IFNs (such as IFN-λ1 and IFN-λ2/3,); tumor necrosis factors (TNF), such as TNF-α, TNF-β, 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 .

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 µm, preferably from 30 to 150 µm, more preferably from 50 to 100 µm. For example, the particles have a diameter of about 80 µm. 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 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), poly(propylene 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 µm, preferably from 100 nm to 10 µm, more preferably from 1 µm 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 µm to 50 µm, especially 5 µm to 20 µm. 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 Fe₃O₄.

The method for moving a payload particle

The system according to 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

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 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, 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.

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;

In specific embodiments, the kit further comprises a capture particle or material for producing a capture particle.
The embodiments, features and examples described herein for the other aspects, especially for the system, also likewise apply to the kit 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.

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 µm or less, preferably 100 µm or less, and more preferably 20 µm 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 Fe₃O₄, Nd, Ni, Co, Nd₂Fe₁₄B, 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 µm from the positioning particle, preferably from 20 to 1500 µm, more preferably from 100 to 500 µm.
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 µm, preferably from 1 to 90 µm.
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 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 any one of embodiments 25 to 27, wherein the material of the payload particle includes a synthetic polymer and/or a natural polymer for cell-encapsulation.
Embodiment 37. The system according to embodiment 36, wherein the synthetic polymer is selected from the group consisting of poly(ethylene glycol) (PEG), poly(ethylene oxide) (PEO), poly(vinyl alcohol) (PVA), poly(propylene fumarate) (PPF), poly(acrylic acid) (PAA), poly(acrylic acid-co-acrylamide) (PAAAm), polyacrylamide (PAAm), polylactic acid, polyglycolic acid, and polyoxazoline.
Embodiment 38. The system according to embodiment 36, wherein the natural polymer is selected from the group consisting of agarose, chitosan, collagen, and alginate.
Embodiment 39. The system according to any one of embodiments 1 to 38, 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 40. The system according to any one of embodiments 1 to 39, wherein the system further comprises a capture particle positioned within the microfluidic channel.
Embodiment 41. The system according to embodiment 40, wherein the capture particle is positioned adjacent to or in the vicinity of the payload particle.
Embodiment 42. The system according to embodiment 40 or 41, wherein the capture particle and the payload particle are positioned within the microfluidic channel at a distance of 500 µm or less, preferably 100 µm or less, more preferably 20 µm or less; most preferably the capture particle and the payload particle are in contact with each other.
Embodiment 43. The system according to any one of embodiments 40 to 42, wherein the capture particle is moved together with the payload particle.
Embodiment 44. The system according to any one of embodiments 40 to 43, wherein the capture particle is capable of capturing analytes released from the payload of the payload particle.
Embodiment 45. The system according to any one of embodiments 1 to 39, wherein the positioning particle is capable of capturing analytes released from the payload of the payload particle.
Embodiment 46. The system according to embodiment 44 or 45, 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 47. The system according to any one of embodiments 44 to 46, wherein the analytes are captured by capture molecules attached to or associated with the capture particle or positioning particle.
Embodiment 48. The system according to embodiment 47, wherein the capture molecules are selected from the group consisting of antibodies, antibody fragments and aptamers.
Embodiment 49. The system according to any one of embodiments 1 to 48, wherein the positioning particle, the payload particle and/or the capture particle are elastic particles.
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 hydrogel particles.
Embodiment 51. The system according to embodiment 50, wherein the hydrogel particles are composed of a hydrogel matrix.
Embodiment 52. The system according to embodiment 51, wherein the hydrogel matrix comprises a synthetic polymer or a natural polymer.
Embodiment 53. The system according to embodiment 52, wherein the synthetic polymer is selected from the group consisting of poly(ethylene glycol) (PEG), poly(ethylene oxide) (PEO), poly(vinyl alcohol) (PVA), poly(propylene fumarate) (PPF), poly(acrylic acid) (PAA), poly(acrylic acid-co-acrylamide) (PAAAm), polyacrylamide (PAAm), polylactic acid, polyglycolic acid, and polyoxazoline.
Embodiment 54. The system according to embodiment 52, wherein the natural polymer is selected from the group consisting of agarose, chitosan, collagen, and alginate.
Embodiment 55. The system according to any one of embodiments 51 to 54, wherein the hydrogel matrix has a stiffness represented by Young's moduli (E) in the range of from 300 to 5400 Pa.
Embodiment 56. The system according to embodiment 51, wherein the hydrogel matrix comprises poly (acrylic acid) polymers and/or agarose.
Embodiment 57. The system according to any one of embodiments 1 to 56, wherein the particles are substantially spherical.
Embodiment 58. The system according to any one of embodiments 1 to 57, wherein the particles have a diameter in the range of from 1 to 200 µm, preferably from 30 to 150 µm, more preferably from 50 to 100 µm.
Embodiment 59. The system according to any one of embodiments 1 to 58, wherein the positioning particle comprises nanoparticles.
Embodiment 60. The system according to embodiment 59, wherein the nanoparticles have a diameter in the range of from 1 nm to 100 µm, preferably from 100 nm to 10 µm, more preferably from 1 µm to 10 nm.
Embodiment 61. The system according to embodiment 59 or 60, wherein the nanoparticles comprise gold, silver, silica, quantum dots, or Fe₃O₄.
Embodiment 62. The system according to any one of embodiments 59 to 61, wherein the nanoparticles are loaded with basic cargo such as NaOH, or with acidic cargo such as HCI or acetic acid.
Embodiment 63. The system according to any one of embodiments 59 to 62, 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 64. The system according to any one of embodiments 1 to 63, wherein the system comprises a means for applying a microfluidic flow through the microfluidic channel.
Embodiment 65. The system according to any one of embodiments 1 to 64, 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 66. The system according to any one of embodiments 1 to 65, wherein the microfluidic channel has a diameter in the range of from 1 to 500 µm, preferably from 30 to 200 µm, more preferably from 50 to 120 µm.
Embodiment 67. The system according to any one of embodiments 1 to 66, wherein 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.
Embodiment 68. The system according to any one of embodiments 1 to 67, wherein the microfluidic channel is part of a microfluidic chip.
Embodiment 69. The system according to any one of embodiments 1 to 68, 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 70. The system according to embodiment 69, 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 71. The system according to any one of embodiments 1 to 70, wherein the positioning particle is not actuated by a microfluidic flow.
Embodiment 72. A method for moving a payload particle in a microfluidic channel, comprising the steps of
1. (i) providing a payload particle and a positioning particle in a microfluidic channel;
2. (ii) initiating movement of the payload particle by actuating the positioning particle.
Embodiment 73. The method according to embodiment 72, having one or more of the features of the system as defined in embodiments 1 to 71.
Embodiment 74. The method according to embodiment 72 or 73, further comprising the step of applying a microfluidic flow to the microfluidic channel.
Embodiment 75. The method according to embodiment 74, wherein the microfluidic flow is applied to the microfluidic channel during steps (i) and (ii).
Embodiment 76. The method according to embodiment 74 or 75, wherein a constant microfluidic flow is applied to the microfluidic channel.
Embodiment 77. The method according to any one of embodiments 72 to 76, wherein the payload particle is moved to or from a position for analyzing and/or manipulating the payload of the payload particle.
Embodiment 78. The method according to any one of embodiments 72 to 77, 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 79. The method according to embodiment 78, wherein the payload particle is moved by the movement of the positioning particle.
Embodiment 80. The method according to embodiment 78 or 79, 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 81. The method according to embodiment 78 or 79, 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 82. The method according to any one of embodiments 78 to 81, 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 83. The method according to any one of embodiments 72 to 77, wherein the positioning particle is responsive to light and wherein actuating the positioning particle includes applying light to the positioning particle.
Embodiment 84. The method according to embodiment 83, wherein the light causes the positioning particle to
1. (i) shrink, allowing a microfluidic flow applied to the microfluidic channel to pass and/or move the positioning particle, and move the payload particle;
2. (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
3. (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 85. The method according to embodiment 83 or 84, wherein actuating the positioning particle in step (ii) includes moving and/or switching on or off of a light source.
Embodiment 86. A kit of parts, comprising
1. (i) a payload particle or material for producing a payload particle; and
2. (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 87. The kit of parts according to embodiment 86, further comprising a capture particle or material for producing a capture particle.
Embodiment 88. The kit of parts according to embodiment 86 or 87, 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 71.
Embodiment 89. 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 90. The use according to embodiment 89, having one or more of the features of the system as defined in embodiments 1 to 71.

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 bead trap (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 bead trap (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 bead trap (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 bead trap (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 bead trap (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 bead trap (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 bead trap (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 bead trap (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 bead trap (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 bead trap (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 bead trap (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 bead trap (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 bead trap (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 bead trap (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 bead trap (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 bead trap (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 bead trap (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 bead trap (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 bead trap (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 bead trap (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 bead trap (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 bead trap (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 bead trap (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 bead trap (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 bead trap (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 bead trap (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 bead trap (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 bead trap (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 bead trap (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 bead trap (11) (lower illustration).

Reference signs

1	microfluidic channel	7	light source
2	payload particle	8	microfluidic flow
3	payload	9	gas bubble
4	positioning particle	10	capture particle
5	source of a magnetic field	11	microfluidic bead trap
6	magnetic force

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 µm. 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, 200µL 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 µL of sterile filtered PBS and 100µL PFO to the particle solution. The aqueous phase was filtered by a 100 µm 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 µL 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 µm 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.

Claims

A system comprising a microfluidic channel (1) and positioned within said microfluidic channel (1) a payload particle (2) and a positioning particle (4);
wherein the positioning particle (4) is capable of being actuated; wherein actuating the positioning particle (4) initiates movement of the payload particle (2).
The system according to claim 1, wherein the positioning particle (4) and the payload particle (2) are adjacent to each other or in the vicinity of each other in the microfluidic channel (1).
The system according to claim 1 or 2, wherein the positioning particle (4) in a resting state and/or the payload particle (2) are fixed at their positions in the microfluidic channel (1), and the positioning particle (4) in its resting state preferably prevents the payload particle (2) from moving.
The system according to any one of claims 1 to 3, wherein the positioning particle (4) is responsive to a magnetic field and wherein actuating the positioning particle (4) includes moving the positioning particle (4) within the microfluidic channel (1) using a magnetic field, wherein the payload particle (2) is moved by the movement of the positioning particle (4), and wherein the system optionally further comprises a magnet as source of the magnetic field (5).
The system according to claim 4, wherein when the positioning particle (4) is moved towards the payload particle (2), the payload particle (2) is pushed in the direction of the movement of the positioning particle (4), either by direct contact to the positioning particle (4), or by the increased pressure in the fluid between the payload particle (2) and the positioning particle (4) caused by the movement of the positioning particle (4).
The system according to any one of claims 1 to 5, wherein the positioning particle (4) is responsive to light and wherein actuating the positioning particle (4) includes applying light to the positioning particle (4), wherein the system optionally further comprises a light source (7).
The system according to claim 6, wherein the light causes the positioning particle (4)
(a) to shrink, allowing a microfluidic flow (8) applied to the microfluidic channel (1) to pass and/or move the positioning particle (4), and move the payload particle (2); or

(b) to swell, whereby the payload particle (2) is pushed away from the positioning particle (4), either by direct contact to the positioning particle (4), or by the increased pressure in the fluid between the payload particle (2) and the positioning particle (4) caused by the swelling of the positioning particle (4).
The system according to any one of claims 1 to 7, wherein the payload particle (2) comprises a payload of interest (3), in particular one or more biological cells, especially one cell or a cell pair.
The system according to any one of claims 1 to 8, wherein the positioning particle (4) is capable of capturing analytes released from the payload (3) of the payload particle (2).
The system according to any one of claims 1 to 8, wherein the system further comprises a capture particle (10) positioned within the microfluidic channel (1), in particular adjacent to or in the vicinity of the payload particle (2); wherein the capture particle (10) is capable of capturing analytes released from the payload (3) of the payload particle (2).
The system according to any one of claims 1 to 10, wherein the positioning particle (4), the payload particle (2) and/or the capture particle (10) have one or more of the following characteristics:
(a) they are elastic particles; and/or

(b) they are composed of a hydrogel matrix which preferably comprises poly(acrylic acid) polymers and/or agarose; and/or

(c) they are substantially spherical.
The system according to any one of claims 1 to 11, wherein the positioning particle (4) comprises nanoparticles, in particular magnetic nanoparticles.
The system according to any one of claims 1 to 12, wherein the system comprises a means for applying a microfluidic flow (8) through the microfluidic channel (1).
The system according to any one of claims 1 to 13, wherein the microfluidic channel (1) is part of a microfluidic chip.
A method for moving a payload particle (2) in a microfluidic channel (1), comprising the steps of
(i) providing a payload particle (2) and a positioning particle (4) in a microfluidic channel (1), wherein the positioning particle (4) and the payload particle (2) are adjacent to each other or in the vicinity of each other in the microfluidic channel;

(ii) initiating movement of the payload particle (2) by actuating the positioning particle (4);
wherein a constant flow is applied to the microfluidic channel throughout the method.
The method according to claim 15, wherein the positioning particle (4) is responsive to a magnetic field, and wherein actuating the positioning particle (4) in step (ii) includes moving the positioning particle (4) using a magnetic field, wherein the payload particle (2) is moved by the movement of the positioning particle (4).
A kit of parts, comprising
(i) a payload particle (2) or material for producing a payload particle (2); and

(ii) and a positioning particle (4) or material for producing a positioning particle (4);
wherein the payload particle (2) and the positioning particle (4) are for use in a microfluidic channel (1); wherein the positioning particle (4) is capable of being actuated; and wherein actuating the positioning particle (4) initiates movement of the payload particle (2).
Use of a positioning particle (4) for initiating movement of a payload particle (2) in a microfluidic channel (1); wherein the positioning particle (4) is capable of being actuated; and wherein actuating the positioning particle (4) initiates movement of the payload particle (2).