EP4122599A1 - Système de commande de mouvement de microparticules - Google Patents

Système de commande de mouvement de microparticules Download PDF

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Publication number
EP4122599A1
EP4122599A1 EP21186702.3A EP21186702A EP4122599A1 EP 4122599 A1 EP4122599 A1 EP 4122599A1 EP 21186702 A EP21186702 A EP 21186702A EP 4122599 A1 EP4122599 A1 EP 4122599A1
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EP
European Patent Office
Prior art keywords
particle
positioning
payload
microfluidic channel
microfluidic
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP21186702.3A
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German (de)
English (en)
Inventor
Robert WEINGARTEN
Sebastian BÜHREN
Hans KLEINE-BRÜGGENEY
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Evorion Biotechnologies GmbH
Original Assignee
Evorion Biotechnologies GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Evorion Biotechnologies GmbH filed Critical Evorion Biotechnologies GmbH
Priority to EP21186702.3A priority Critical patent/EP4122599A1/fr
Priority to PCT/EP2022/070377 priority patent/WO2023001898A1/fr
Publication of EP4122599A1 publication Critical patent/EP4122599A1/fr
Pending legal-status Critical Current

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502715—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/50273—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the means or forces applied to move the fluids
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502761—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • B01L2200/0668—Trapping microscopic beads
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/043—Moving fluids with specific forces or mechanical means specific forces magnetic forces
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/06—Valves, specific forms thereof
    • B01L2400/0633—Valves, specific forms thereof with moving parts
    • B01L2400/0672—Swellable plugs
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/06—Valves, specific forms thereof
    • B01L2400/0677—Valves, specific forms thereof phase change valves; Meltable, freezing, dissolvable plugs; Destructible barriers

Definitions

  • the present invention generally concerns single cell analysis and similar methods for analyzing particles in microscale.
  • 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.
  • Fluid C1 platform 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.
  • 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.
  • the present inventors have developed a system for controlling the positioning and movement of a particle for microanalysis.
  • 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.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • the present invention provides a method for moving a payload particle in a microfluidic channel, comprising the steps of
  • 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 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 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.
  • a second particle the positioning particle
  • movement of the payload particle is initiated.
  • the payload particle may be pushed or pulled by positioning particle which is actively moved through movement of a magnetic field.
  • the payload particle may be pushed by swelling the positioning particle directly adjacent to the payload particle.
  • 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.
  • this system only the positioning particle is manipulated, e.g.
  • the payload particle comprising the product of interest is not affected by any of these control mechanisms, which therefore do not disturb the analysis.
  • microfluidic system The microfluidic system
  • 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.
  • the microfluidic channel(s) has a diameter in the range of from 1 to 500 ⁇ m, preferably from 30 to 200 ⁇ m, more preferably from 50 to 120 ⁇ m.
  • the microfluidic channel(s) may have a diameter of about 70 to 100 ⁇ m.
  • 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.
  • the microfluidic channel(s) may have a breadth of about 100 ⁇ m and a height of about 80 ⁇ m.
  • 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.
  • a microfluidic flow may be achieved using capillary forces.
  • 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.
  • one payload particle and one positioning particle form a pair, wherein actuating the positioning particle initiates movement of the paired payload particle.
  • positioning particle and payload particle especially refer to the particles of a pair of positioning and payload particle.
  • the system comprises a plurality of pairs positioned within said microfluidic channel, wherein each pair comprising exactly one payload particle and one positioning particle.
  • 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.
  • the positioning particle and the payload particle are adjacent to each other or in the vicinity of each other in the microfluidic channel.
  • the positioning particle and the payload particle are positioned within the microfluidic channel at a distance of 200 ⁇ m or less, preferably 100 ⁇ m or less, and more preferably 20 ⁇ m or less.
  • the positioning particle and the payload particle are in contact with each other.
  • 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.
  • the force is not the microfluidic flow within the microfluidic channel or system.
  • the force is applied from outside of the microfluidic channel.
  • the positioning particle is not actuated by a microfluidic flow.
  • the positioning particle 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%.
  • the positioning particle in its resting state is fixed at its position in the microfluidic channel.
  • the positioning particle is wedged in the microfluidic channel due to its size.
  • the positioning particle in its resting state is not moved by a microfluidic flow applied to the system or the microfluidic channel.
  • the payload particle in its resting state is fixed at its position in the microfluidic channel.
  • the payload particle is wedged in the microfluidic channel due to its size.
  • 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.
  • the movement of the payload particle which is initiated is in the direction of the microfluidic flow.
  • the movement of the payload particle which is initiated is against the direction of the microfluidic flow.
  • no microfluidic flow is applied to the microfluidic channel.
  • the positioning particle is used for controlling the movement and position of the payload particle.
  • the positioning particle is capable of being actuated.
  • 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.
  • the reaction of the positioning particle to the external force leads to a movement of the payload particle.
  • 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.
  • the positioning particle is responsive to a magnetic field.
  • 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.
  • the positioning particle is moved towards the payload particle.
  • 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.
  • 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 and/or a change of microfluidic flow that is initiated due to the actuation of the positioning particle.
  • 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.
  • the positioning particle comprises magnetic nanoparticles.
  • the magnetic material, especially the magnetic nanoparticles may be ferromagnetic, ferrimagnetic, paramagnetic, superparamagnetic, or diamagnetic.
  • the magnetic material, especially the magnetic nanoparticles has a high uniaxial magnetocrystalline anisotropy.
  • the magnetic material, especially the magnetic nanoparticles comprise material selected from Fe 3 O 4 , Nd, Ni, Co, Nd 2 Fe 14 B, and tetracyanoquinodimethane, or a combination thereof.
  • the magnetic material, especially the magnetic nanoparticles may consist of such material.
  • the magnetic material, especially the magnetic nanoparticles is coated, for example with polyaniline.
  • the system further comprises a magnet as source of the magnetic field.
  • exemplary magnets include permanent magnets and electromagnets.
  • the source of the magnetic field may be a neodymium magnet.
  • 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 ⁇ m from the positioning particle, preferably from 20 to 1500 ⁇ m, more preferably from 100 to 500 ⁇ m.
  • 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.
  • the source of the magnetic field can be moved relative to the microfluidic channel and/or turned on and off.
  • 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.
  • 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.
  • 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.
  • the positioning particle is responsive to light.
  • 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.
  • 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.
  • shrinking of the positioning particle induces a local change of hydrodynamic resistance and thus, a change in the microfluidic flow.
  • 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.
  • 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.
  • the positioning particle 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.
  • 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.
  • the light induces hydrolysis, protonation or deprotonation of chemical groups within the material of the positioning particle.
  • complementary charged chemical groups are generated, which decreases electrostatic repulsion between charged groups and/or decreases osmotic pressure.
  • 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.
  • 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.
  • the light induces hydrolysis, protonation or deprotonation of chemical groups within the material of the positioning particle.
  • chemical groups with the same charge are generated, which increases electrostatic repulsion between charged groups and/or increases osmotic pressure.
  • 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.
  • the formed bubble has a diameter in the range of from 1 to 500 ⁇ m, preferably from 1 to 90 ⁇ m.
  • Applying light to the positioning particle may in particular cause a local change of characteristics of the positioning particle.
  • 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:
  • 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.
  • 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.
  • the light source is capable of specifically illuminating the positioning particle.
  • 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 ⁇ m, preferably 1 to 10 ⁇ m, such as about 3 ⁇ m.
  • 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.
  • 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 may be any suitable particle for use in microfluidic systems.
  • the payload particle itself or its payload is an object of analysis performed using the system.
  • 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.
  • the payload is encapsulated within the payload particle.
  • the payload of interest is a biological cell.
  • the payload may be one or more than one cell.
  • 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.
  • 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.
  • 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.
  • the material of the payload particle comprises a natural polymer selected from the group consisting of agarose, chitosan, collagen, and alginate.
  • 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.
  • the matrix of the payload particle has a stiffness represented by Young's moduli (E) in the range of from 300 to 5400 Pa.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 invention overcomes significant technical challenges thereby making microfluidic cell culture procedures accessible for downstream analysis such as next-generation sequencing.
  • 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.
  • 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.
  • 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.
  • the system further comprises a capture particle positioned within the microfluidic channel.
  • the capture particle is positioned adjacent to or in the vicinity of the payload particle.
  • 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.
  • the capture particle and the payload particle are positioned within the microfluidic channel at a distance of 200 ⁇ m or less, preferably 100 ⁇ m or less, and more preferably 20 ⁇ m 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.
  • the capture particle is capable of capturing analytes released from the payload of the payload particle.
  • the positioning particle is capable of capturing analytes released from the payload of the payload particle.
  • 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.
  • 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.
  • the analytes preferably are selected from the group comprising peptides, polypeptides, proteins, carbohydrates, nucleic acids, small organic molecules and lipids.
  • 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).
  • the analytes are selected from the group consisting of interleukins (ILs), including IL-1 ⁇ , IL-1 ⁇ , 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-36 ⁇ , IL-36 ⁇ , IL-36 ⁇ , IL-37, IL-1Ra, IL-36Ra and IL-38; interferons (INFs), including type I IFNs (such as IFN- ⁇ (further classified into 13 different sub
  • Particular analytes of interest include EGF, VEGF, CCL2, CCL5, IL-6 and IL-10.
  • growth factors such as EGF and VEGF are analyzed
  • chemokines such as CCL2 and CCL5 are analyzed
  • interleukins such as IL-6 and IL-10 are analyzed.
  • Suitable analytes and capture molecules and their integration into hydrogel particles are described, for example, in WO 2020/183015 A1 .
  • the particles of the system generally may be any type of particles as long as they are capable of exerting the functions described herein.
  • the particles of the system are elastic particles.
  • the particles have a stiffness represented by Young's moduli (E) in the range of from 300 to 5400 Pa.
  • the particles of the system are substantially spherical.
  • the particles have a diameter in the range of from 1 to 200 ⁇ m, preferably from 30 to 150 ⁇ m, more preferably from 50 to 100 ⁇ m.
  • the particles have a diameter of about 80 ⁇ m.
  • the particles have a diameter which is similar to the diameter of the microfluidic channel.
  • the diameter of the particles of the system is within +/-10% of the diameter of the microfluidic channel, especially within +/- 5%.
  • the particles of the system are hydrogel particles.
  • a hydrogel particle is composed of a hydrogel matrix.
  • the hydrogel matrix may comprise a synthetic polymer or a natural polymer.
  • 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.
  • the hydrogel matrix comprises a natural polymer selected from the group consisting of agarose, chitosan, collagen, and alginate.
  • the hydrogel matrix comprises a mixture of at least two different polymers. Suitable hydrogel matrices are disclosed, for example, in WO 2019/048714 A2 .
  • the hydrogel matrix comprises poly (acrylic acid) polymers and/or agarose.
  • one or more of the particles of the system comprises nanoparticles.
  • 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.
  • the nanoparticles have a diameter in the range of from 1 nm to 100 ⁇ m, preferably from 100 nm to 10 ⁇ m, more preferably from 1 ⁇ m to 10 nm.
  • the diameter of the nanoparticles in particular refers to their largest diameter.
  • the nanoparticles are bound to the particle of the system with an equilibrium dissociation constant of less than 10 -12 M.
  • the positioning particle comprises only one nanoparticle.
  • the nanoparticle preferably has a size in the range of 1 ⁇ m to 50 ⁇ m, especially 5 ⁇ m to 20 ⁇ m. 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.
  • the nanoparticles are used for rendering the positioning particle actuatable.
  • 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.
  • 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.
  • the nanoparticles may be used for improving identification of the particles, for heating the particles, and/or for plasmonic effects.
  • the nanoparticles comprise of gold and/ or silver to use plasmonic principles.
  • the nanoparticles comprise a material selected from the group consisting of gold, silver, silica, quantum dots, and Fe 3 O 4 .
  • 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.
  • 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 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.
  • the payload particle is moved to or from a position for analyzing and/or manipulating the payload of the payload particle.
  • 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).
  • 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.
  • actuating the positioning particle is achieved by using a magnetic field.
  • actuating the positioning particle in step (ii) includes moving the positioning particle using a magnetic field.
  • the positioning particle is responsive to a magnetic field.
  • 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.
  • 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.
  • 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.
  • actuating the positioning particle is achieved by applying light to the positioning particle.
  • the positioning particle is responsive to light.
  • 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
  • Kits comprising the particles
  • 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
  • the kit further comprises a capture particle or material for producing a capture particle.
  • the material for producing the positioning particle and/or the payload particle and/or the capture particle may be reagents for forming the particles.
  • the material comprises a hydrogel or reagents for forming a hydrogel.
  • the material for producing the positioning particles may comprise suitable nanoparticles.
  • 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.
  • 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.
  • 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”.
  • 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.
  • Droplet formation was performed in a microfluidic flow-focusing device with a channel width of 80 ⁇ m.
  • 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, 200 ⁇ L 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 ⁇ L of sterile filtered PBS and 100 ⁇ L PFO to the particle solution. The aqueous phase was filtered by a 100 ⁇ m mesh filter (Sysmex, Kobe, Japan).
  • 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 ⁇ L of the prepared hydrogel/particle mixture.
  • 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.
  • 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 ⁇ m 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.
  • 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.

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EP21186702.3A 2021-07-20 2021-07-20 Système de commande de mouvement de microparticules Pending EP4122599A1 (fr)

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EP21186702.3A EP4122599A1 (fr) 2021-07-20 2021-07-20 Système de commande de mouvement de microparticules
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