WO2023186984A2 - Puce, dispositif et procédé d'analyse de particules en suspension - Google Patents

Puce, dispositif et procédé d'analyse de particules en suspension Download PDF

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Publication number
WO2023186984A2
WO2023186984A2 PCT/EP2023/058145 EP2023058145W WO2023186984A2 WO 2023186984 A2 WO2023186984 A2 WO 2023186984A2 EP 2023058145 W EP2023058145 W EP 2023058145W WO 2023186984 A2 WO2023186984 A2 WO 2023186984A2
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WO
WIPO (PCT)
Prior art keywords
chip
microchannel
base body
suspended particles
reservoir
Prior art date
Application number
PCT/EP2023/058145
Other languages
German (de)
English (en)
Other versions
WO2023186984A3 (fr
Inventor
Stephan Quint
Original Assignee
Cysmic 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
Priority claimed from DE102022107729.2A external-priority patent/DE102022107729A1/de
Application filed by Cysmic GmbH filed Critical Cysmic GmbH
Publication of WO2023186984A2 publication Critical patent/WO2023186984A2/fr
Publication of WO2023186984A3 publication Critical patent/WO2023186984A3/fr

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Classifications

    • 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
    • B01L3/502707—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 manufacture of the container or its components
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1484—Optical investigation techniques, e.g. flow cytometry microstructural devices
    • 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
    • B01L2200/02—Adapting objects or devices to another
    • B01L2200/025—Align devices or objects to ensure defined positions relative to each other
    • 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
    • B01L2200/02—Adapting objects or devices to another
    • B01L2200/026—Fluid interfacing between devices or objects, e.g. connectors, inlet details
    • B01L2200/027—Fluid interfacing between devices or objects, e.g. connectors, inlet details for microfluidic devices
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0681—Filter
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions
    • 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
    • B01L2400/0487—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics
    • 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
    • B01L2400/08—Regulating or influencing the flow resistance
    • B01L2400/084—Passive control of flow resistance
    • B01L2400/086—Passive control of flow resistance using baffles or other fixed flow obstructions
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/01—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials specially adapted for biological cells, e.g. blood cells
    • G01N2015/012—Red blood cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N2015/1006—Investigating individual particles for cytology

Definitions

  • Chip device and method for analyzing suspended particles
  • the invention relates to the measurement of suspended particles, in particular red blood cells, which move either with or in the suspension.
  • the present invention is concerned with measuring the properties of moving suspended particles, whereby the properties of the particles can be influenced by the movement of the particles.
  • the invention relates to a chip for the analysis of suspended particles according to patent claim 1, the use of a chip for the analysis of suspended particles according to patent claim 18 and a method for producing a chip according to patent claims 19 and 26.
  • the invention further relates to a device for analyzing suspended particles according to patent claim 27 and the use of the device according to patent claim 37.
  • the invention also relates to a system for analyzing suspended particles according to patent claim 38 and a method for analyzing suspended particles according to patent claim 41.
  • the underlying object of the invention is a chip for the analysis of suspended particles, the use of a chip for the analysis of suspended particles, a method for producing a chip, a device for analysis suspended particles to provide the use of a device, a system for analyzing suspended particles, and a method for analyzing suspended particles, which enable easy-to-handle, rapid and, in particular, on-site analysis of suspended particles, in particular red blood cells.
  • a chip according to the invention is used to analyze suspended particles and in particular to analyze red blood cells.
  • the chip comprises a base body, wherein the base body has at least one microchannel for the passage of the suspended particles.
  • the at least one microchannel in particular has a length (along the channel direction/x-direction) of 1 cm to 10 cm.
  • the microchannel in particular has a width (transverse to the channel direction/y direction) of 5 pm to 15 pm and a height (in the channel vertical direction/z direction) of 5 pm to 15 pm height.
  • the at least one microchannel is rectangular, semicircular or round in cross section or trapezoidal or rectangular with round corners.
  • the microchannel is approximately the size of red blood cells, which usually have a diameter of up to 12 pm (for humans and many animal species) in the resting state.
  • Such a chip is used to imitate the flow conditions in capillaries, such as blood vessels.
  • a chip can be used in particular for the analysis of red blood cells, since degradation of the blood cells or diseases can be concluded based on the shape of the red blood cells, the flow speed of the blood cells and the position of the red blood cells in the microchannel.
  • the chip is particularly cost-effective and can be produced on a large scale and can be used for the individual examination of suspended particles.
  • the chip is practical and easy to use. In particular, it is an exchange product which is only used to analyze a sample of suspended particles.
  • the chip also enables the analysis of individual particles, such as individual red blood cells.
  • the base body has several, in particular parallel (or in a relative Angles of +/- 10 degrees to each other), micro channels.
  • the base body has between 1 and 2000 parallel microchannels.
  • the advantage of multiple microchannels arises in conjunction with a device for analyzing suspended particles. When the manufacturing tolerances are exhausted, there is always at least one microchannel in the field of view of an optical unit (in particular lens and camera), which is used to observe the suspended particles.
  • an xy adjustable table can be dispensed with when analyzing particles flowing through the chip using a device mentioned above.
  • the plurality of microchannels are connected in a flow-conducting manner to a common inlet, so that the suspension flows uniformly from the inlet into the plurality of microchannels.
  • the several microchannels are connected to a common outlet in a flow-conducting manner.
  • the base body of the chip is in particular made of plastic, ceramic or glass. Many plastics are inexpensive, robust and light. For the present application, the material should be transparent to light in the visible wavelength range and, if necessary, in the UV range.
  • the plastics used are in particular elastomers such as TPU, TPE, PDMS (silicone), thermoplastics such as PC, PMMA, COC, COP, PA, PP, PVCPE, PET, PETG, PLA or thermosets such as PUR. or derivatives or mixtures thereof in question.
  • the at least one microchannel in the base body is in particular a microchannel that is open on one side and has a particularly U-shaped cross section, wherein the at least one microchannel is covered by a cover element. Viewed in cross section, the microchannel is then surrounded by the base body on three sides and by the cover element on one side. The cover element and the base body are connected to one another in such a way that the at least one microchannel is sealed off from the environment.
  • the cover element is in particular made of glass.
  • the cover element like the base body, is made of ceramic or plastic, such as TPU, TPE, PDMS, PC, PMMA, COC, COP, PA, PP, PVC, PE, PET, PETG, PLA, PUR derivatives or mixtures thereof.
  • the cover element preferably has a thickness between 50 pm and 1000 pm.
  • the cover element is in particular also transparent to light in the visible spectrum and possibly in the UV spectrum and enables an optical unit (lens and camera) to have as uninfluenced a view as possible of the at least one microchannel and suspended particles flowing therein.
  • the base body made of plastic and the cover element are in particular firmly connected to one another, preferably by means of bonding, in particular by means of so-called plasma activated bonding, UV activated bonding, solvent bonding, diffusion bonding or by adhesive (glue) .
  • the plastics mentioned above are each suitable for different bonding processes.
  • the at least one microchannel of the base body and the cover element can also be designed completely as a cavity in the base body.
  • the base body has at least one reservoir for storing the suspended particles.
  • the reservoir is in particular designed in one piece with the base body.
  • the base body has a first reservoir and a second reservoir, the first reservoir serving to store suspended particles that have not yet flowed through a microchannel and the second reservoir serving to store suspended particles that have already flowed through a microchannel and have already been analyzed.
  • the first reservoir and the second reservoir are preferably designed identically.
  • the reservoir can be designed as a simple cavity on the base body, so that one or more drops of the suspended liquid can be easily added there.
  • a riser pipe ends in the at least one reservoir, the riser pipe establishing a flow-conducting connection between the reservoir and the at least one microchannel and the riser pipe ending above the bottom of the reservoir.
  • a funnel-shaped opening is formed at the bottom of the reservoir, which is in a flow-conducting connection with the at least one microchannel.
  • At least one filter is arranged at at least one end of the at least one microchannel.
  • the filter is intended to prevent any impurities (and in the case of blood analysis, any larger blood components such as leukocytes) from entering the microchannel and clogging it.
  • a filter is arranged at both ends of a microchannel.
  • the chip can be inserted into a device for analysis in any direction, or flow can flow through the chip in any direction, which prevents possible incorrect use of the chip.
  • several elements arranged next to one another and/or one behind the other can serve as filters, each of which is arranged in a supply area to the microchannels and whose distance from one another is chosen to be so small that larger particles cannot pass through them.
  • the chip is mirror-symmetrical with respect to a plane (yz plane) perpendicular to the at least one microchannel.
  • the chip can then be rotated 180° around its vertical direction without this affecting the analysis. This also prevents possible incorrect use of the chip during analysis.
  • the chip has two identical reservoirs and possibly also filters on both sides of the at least one microchannel.
  • the at least one microchannel can have a narrowing.
  • the narrowing can be formed by at least one projection projecting into the microchannel. If the base body has several microchannels, this is the case at least one of these microchannels has a narrowing.
  • Microchannels with a narrowing and without a narrowing are preferably arranged alternately. This in turn has the advantage that there is always a channel with a narrowing and a channel without a narrowing in the field of view of an optical unit.
  • the narrowing can, for example, simulate a narrowed bloodstream (stenosis).
  • the behavior of the suspended particles, especially red blood cells, in a narrowed microchannel can provide information about whether or not they are retained in narrowed bloodstreams or irreversibly damaged.
  • the base body is designed in at least two parts and comprises a channel element and a carrier element.
  • the channel element has at least one microchannel.
  • the carrier element is arranged on a surface of the channel element facing away from the at least one microchannel.
  • the two-part design of the base body described above is particularly suitable for channel elements that are embedded in elastomers (e.g. silicone) that have a comparatively low strength but high transparency and bonding ability.
  • the carrier element can then serve to mechanically stabilize and handle the base body.
  • the carrier element is in particular made of glass, ceramic or plastic such as TPU, TPE, PDMS, PC, PMMA, COC, COP, PA, PP, PVC, PE, PET, PETG, PLA, PUR. Derivatives or mixtures thereof.
  • the carrier element in particular has at least one first reservoir, the reservoir being in flow-conducting connection with an inlet end of the at least one microchannel.
  • the carrier element has in particular a second reservoir, wherein the second reservoir is in flow-conducting connection with an outlet end of the at least one microchannel.
  • the carrier element and the channel element are in particular captively or positively connected to one another in order to ensure the best possible durability of the chip.
  • the positive connection is realized in particular in that at least one connecting section is formed on the surface of the channel element facing the carrier element, the connecting section extending through a corresponding opening in the carrier element extends and engages behind it. If there are several connecting sections, these can be connected to the sections behind them and form a common, flat surface. As explained below, this gripping behind the opening can be realized in particular simply by pressing the carrier element onto the still deformable channel element, so that the material of the channel element swells through the openings in the carrier element. The channel element and the carrier element are then also sealingly connected to one another.
  • the base body has a viewing window.
  • a viewing window is an area of the base body which is, in particular, optically transparent in the visible wavelength range and/or in the UV range. This transparency is achieved both by the material properties of the viewing window and by the surface quality.
  • the viewing window is in particular designed in one piece with the base body.
  • the material of the base body is particularly smooth here so that the light scattering on the surface of the viewing window is as low as possible.
  • the viewing window is manufactured in particular by being molded from a polished surface.
  • the surface of the viewing window is arranged at a distance of 10 pm to 10 mm from the canal base, with a small distance having a positive effect on possible optical distortions and ensuring better imaging.
  • the base body in particular has a recess and has a reduced material thickness in the area of the viewing window. The material thickness is in particular in the range from 100 pm to 5 mm.
  • the suspended particles flowing in the chip are analyzed using transmitted light microscopy, i.e. the illumination takes place from the side of the chip remote from the microchannel and the image recognition takes place on the side of the chip facing the microchannel. Due to the small thickness of the entire chip in the area of the viewing window, the light source can be brought as close as possible to the microchannel and to the optical unit arranged on the other side of the microchannel. Diffraction effects that can arise due to any deformation of the base body generally play a minor role here. The analysis and image evaluation are therefore free of distortions. Overall, the chip enables with Such a viewing window provides particularly good imaging quality of the suspended particles, which also facilitates subsequent image evaluation.
  • both the carrier element and the channel element can have a reduced material thickness in the two-part base body.
  • a recess in the area of the viewing window can in particular be surrounded by the carrier element.
  • the base body can have at least one positioning aid.
  • the base body preferably has several positioning aids distributed around the viewing window.
  • a projection that protrudes from an edge of the viewing window into the area of the viewing window can serve as a positioning aid.
  • Such a positioning aid can be used to place an optical element such as an optical mask, an optical filter, a mirror, a polarizer, a diffuser or generally refractive or diffractive optics or combinations thereof.
  • an optical slit mask can then be arranged in the area of the viewing window, which is useful for image evaluation.
  • the mask can, for example, have translucent (slits) and opaque areas (webs).
  • the optical element can be injected into the base body.
  • the positioning aid can also be designed or arranged on the carrier element in the two-part base body.
  • the base body has an at least partially circumferential collar.
  • the collar extends over the entire circumference of the base body.
  • the collar is particularly important in connection with the device for analyzing the suspended particles.
  • the chip With the at least partially circumferential collar, the chip can be inserted into a positioning receptacle in the device with repeatable accuracy. Furthermore, the chip can be supported on the device via the collar.
  • the latter is particularly advantageous with a channel element made of silicone and with a cover element made of glass, which can crack under pressure. So that the at least one microchannel in the silicone is not deformed, the carrier element has the collar and supports the entire chip in the device against attacking forces.
  • the invention also relates to the use of a chip as described above for analyzing suspended particles, in particular for analyzing suspended red blood cells.
  • the invention also relates to a method for producing a chip for analyzing suspended particles, in particular a chip as described above.
  • the method includes the following process steps: a) providing a structured impression part with at least one elevation for structuring a microchannel, b) inserting the impression part into a mold, c) filling material into the mold to form a base body, d) hardening the base body, e ) Removing the molded base body from the mold, f) Arranging a cover element on a first surface of the base body, which has the at least one microchannel.
  • the impression part has several elongated elevations for structuring several microchannels on the base body.
  • the process for producing the chip is particularly simple and is suitable for producing large quantities of chips for analyzing suspended particles.
  • the shape in particular also includes structures for forming at least one reservoir and/or for forming at least one filter.
  • the base body is produced from plastic by injection molding in step c).
  • the plastic is in particular elastomers such as TPU, TPE, PDMS, thermoplastics such as PC, PMMA, COC, COP, PA, PP, PVC, PE, PET, PETG, PLA, thermosets such as PUR, derivatives or mixtures thereof.
  • Injection molding is a well-known and cost-effective process that enables high throughput.
  • the structured impression part is in particular a plastic, ceramic, glass, silicon or metal part, in particular made of nickel or a nickel alloy. Before step a), the impression part is formed from a structured silicon wafer, a structured metal or ceramic part. The metal part allows the base body to be easily removed and can be reused several times.
  • the cover element and the base body are subjected to a plasma treatment before they are connected.
  • a plasma treatment is a plasma made of nitrogen, oxygen, hydrogen and/or a noble gas.
  • the plasma treatment in particular modifies the uppermost atomic layers of the respective parts, down to a depth of a few 100 nm.
  • This so-called plasma activated bonding is particularly suitable for connecting plastics such as TPE or silicone with glass.
  • the connection is then particularly stable and, above all, sealing.
  • the connection can be established reproducibly and with a reasonable amount of time.
  • cover element and the base body are connected using UV bonding, solvent bonding, diffusion bonding or adhesively (see above).
  • the base body is manufactured in two parts and in step c) a material is filled to form a channel element and then a carrier element is placed on the channel element.
  • the carrier element is placed on the channel element and serves to mechanically stabilize the base body. This is particularly advantageous if the channel element is made of silicone and has low mechanical stability.
  • step e) the connected elements of the carrier element and channel element are then removed.
  • the carrier element is already present as a hardened injection-molded part made of plastic.
  • the support element is prepared accordingly manufactured and then placed as a finished component on the just cast or injection molded channel element.
  • the production of the chip with a two-part base body is particularly simple if the carrier element is placed on the channel element with such pressure that the material of the channel element emerges from at least one opening in the carrier element and engages behind this opening. This means that a positive connection between the channel element and the carrier element is realized directly with the production of the base body, without further process steps or even additional connecting elements.
  • the base body has at least one reservoir, with a flow-conducting connection from the reservoir to the at least one microchannel being produced by piercing the base body.
  • This piercing can be done in particular with a needle or cannula.
  • the base body can have a viewing window.
  • the base body has, in particular, a particularly low material thickness and/or a particularly smooth surface in the area of the viewing window.
  • the smooth surface is created in particular by molding the base body from a polished surface.
  • an optical element can also be injected into the base body.
  • the chip can also be manufactured using additive manufacturing. This is particularly suitable for a one-piece base body, with the at least one microchannel being designed as a cavity in the base body.
  • the invention also relates to a device for analyzing suspended particles.
  • the device comprises an optical unit, in particular a lens and a camera.
  • the objective has a magnification of lOx - lOOx.
  • the camera is designed in particular for high-speed recording and has a frame rate of 100 - 2000 s 1 in particular for the region of interest.
  • the device has an object stage for positioning a chip with at least one microchannel for the flow of the suspended particles. In which Chip that can be placed on the object stage is in particular a chip as described above.
  • the device further comprises a connection element for producing a flow-conducting connection with the at least one chip.
  • the connection element serves in particular to pressurize the at least one reservoir to convey the suspended particles through the at least one microchannel.
  • the device is in particular a so-called table-top device, which is suitable for on-site use for analyzing suspended particles, in particular red blood cells.
  • the device enables the analysis of individual particles or individual cells, such as red blood cells.
  • connection element can be pressed onto a chip in particular by means of spring force. This ensures that the connecting element is pressed onto the chip with a defined pressure and that no too much or too little force is exerted on the chip, in order not to damage the chip on the one hand and to ensure sealing on the other.
  • the connection element has a seal.
  • the object stage of the device in particular has a recess for at least partially accommodating a chip, in particular a chip as described above.
  • the depression in particular has an at least partially circumferential contact edge, against which a corresponding collar of the chip can be brought into contact.
  • the chip can be supported on the contact edge via a collar. The force is then absorbed by the chip solely via the collar that is in contact with the contact edge.
  • the at least one microchannel is not damaged. This is particularly advantageous for chips that comprise a comparatively soft material such as silicone.
  • the device in particular has a heating device.
  • the heating device in particular, all areas for contact with a chip or surrounding a chip can be tempered become.
  • the temperature range ranges from 20°C to 50°C.
  • the object stage or a chip receiving area and/or the connection element can be heated.
  • the object stage and/or the connection element are in particular at least partially made of metal.
  • the optical unit is rotatably mounted.
  • the optical unit can be rotated relative to the z direction. This means that possible tilting of the chip/microchannels relative to the z-axis can be easily compensated for by rotating the optical unit.
  • the rotation of the optical unit takes place automatically, especially after an image analysis. The rotation ensures that the viewed section of at least one microchannel lies horizontally in the focal plane of the lens. If there are several microchannels in a parallel array and the optical unit can be rotated, then 2 mechanical degrees of freedom of the system are already “compensated”: rotation around z and y-shift (x-shift is not critical because of the large length of the microchannels in contrast to the field of view ).
  • the last remaining degree of freedom is the z-direction (focus plane); here, in particular, an additional focusing unit is provided, which is set up to carry out autofocus. It is also conceivable to set the tilting using software via an optimizer, which means that the rotation takes place for as long as possible , until the horizontal features are maximum.
  • a possible rotation of the at least one microchannel around the z-axis can alternatively be eliminated by subsequent image correction.
  • the device is particularly designed to be operated in transmitted light mode. This means that the optical unit and the light source are on different sides of the sample to be analyzed or, in this case, the chip. However, it is also conceivable to design the device for the dark field mode, with the light source then being arranged to the side of the optical unit. A fluorescence mode is also possible in conjunction with a filter.
  • a light source in the violet wavelength range is particularly suitable as a light source for the analysis of suspended particles such as red blood cells.
  • An LED can preferably be used that emits light with a wavelength of 415 nm +/- 30 nm emitted. Red blood cells absorb particularly well in this wavelength range.
  • a white light source or a light source that emits light in the red wavelength range can be used to better detect diffraction effects on the cells or to detect light absorption by the lipids (visualization of the cell membrane). .
  • the light source is designed in particular to emit parallel light beams. Light rays propagating from the light source to the optical unit then propagate as parallel as possible through the chip.
  • the light source is positioned in particular 0.1 mm to 200 mm away from the chip.
  • the device is designed in particular as a so-called inverse device.
  • the optical unit consisting of lens and camera is located, viewed in the direction of gravity, below or within the object stage and below an area for placing a chip.
  • the light source is then arranged above the stage.
  • the optical unit can then be brought particularly close to the suspended particles to be analyzed flowing in the microchannel if a chip with the comparatively thin cover plate or the thin base body there is placed downwards on the device.
  • connection element can then be moved relative to the object stage and the chip arranged thereon and in particular the connection element can be placed on the chip. Thanks to the relative movability, the light source can then be positioned as close as possible to the chip.
  • the device has a conveyor device.
  • the conveying device can be used to exert pressure on the first reservoir with the suspended particles to be analyzed and to convey the suspended particles through the at least one microchannel.
  • the conveying device is connected to a pressure accumulator.
  • the conveyor device can be switched off during an ongoing analysis of suspended cells. The device can then be operated with particularly low vibration and the absorption of suspended particles is possible with high quality.
  • the device is set up to analyze the position of the center of gravity of individual suspended particles relative to a center of at least one microchannel.
  • the center of gravity of the particles should be able to be determined in relation to the y-direction or the width direction of the at least one microchannel. This is achieved in particular by detecting the left and right edges of the at least one microchannel with the optical unit and determining the center line from this.
  • the center of gravity of the individual suspended particles is then determined using image analysis and related to the center line. For example, the deformability of red blood cells can be determined based on the distribution of the center of gravity under given flow conditions.
  • an image evaluation takes place. This is carried out locally or in a higher-level unit - in the cloud.
  • the device has a communication device for wireless or wired communication with a higher-level unit. In this way, recorded images can be transmitted to the higher-level unit and results of the image evaluation can also be received by the device. Communication can take place via l-AN/WLAN or via the mobile phone network (GSM/GPRS, UMTS or LTE).
  • the invention also relates to the use of a device as described above for analyzing suspended particles.
  • the invention further relates to a system for analyzing suspended particles, with a device with an optical unit and an object stage.
  • the device is a device as described above.
  • a chip with at least one microchannel is arranged on the stage.
  • the chip is in particular a chip as described above.
  • the system includes a conveyor device for conveying the suspended particles from a first reservoir through the at least one microchannel to a second reservoir.
  • the optical unit is arranged in such a way that the suspended particles in the at least one microchannel can be viewed by means of the optical unit.
  • the system is preferably integrated into a compact device with a housing, which can be used as a table-top device to save space in various locations.
  • the first reservoir serves in particular to store the suspended particles intended for analysis.
  • the first reservoir is pressurized by the conveying device. For example, there can be a pressure of up to 2 bar in the first reservoir.
  • the suspended particles that have already flowed through the chip are collected in the second reservoir.
  • the system can preferably be used to carry out the analysis of suspended particles fully automatically.
  • the system is designed so that the suspended particles flowing through the chip can flow through the at least one microchannel at different pressures.
  • the system has a proportional valve.
  • the pressure acting on flowing suspended particles also influences the speed of the flowing particles. Based on the speed that occurs at a certain pressure, conclusions can then be drawn about the properties of the particles, in particular about the properties of red blood cells.
  • the pressure can be adjustable by the proportional valve, in particular in a range between 10 mbar and 2 bar. In the case of red blood cells, speeds in the range of approximately 0.1 mm/s to 20 mm/s are then achieved (depending on the channel geometry and channel length used).
  • the system has a gas connection for supplying filtered room air, N2, CO2, O2 and/or other process gases into the suspension.
  • a gas connection for supplying filtered room air, N2, CO2, O2 and/or other process gases into the suspension.
  • oxygenation or deoxygenation it is possible to change the properties of the suspended particles.
  • the suspended particles are suspended in a buffer solution.
  • this can in particular be a physiological buffer solution of PBS or PBS and BSA or PBS and HSA or Tyrode or a mixture thereof.
  • the osmolarity and/or the pH value of the respective buffer can be changed in order to view the measurement under different conditions.
  • the invention further relates to a method for analyzing suspended particles, in particular with a system as described above.
  • the suspended particles are guided through a chip (in particular a chip as described above) and at least one microchannel formed thereon.
  • the suspended particles flowing through are recorded with a camera and the suspension is analyzed using image evaluation.
  • a user must in particular place at least one drop of the suspended particles into a first reservoir of a chip with a microchannel as described above; in particular, the particles can be suspended in a physiological buffer solution.
  • a cell concentration of less than 5% in a buffer solution is particularly desirable. This ensures that the blood cells flow through the at least one microchannel individually and not in too high a concentration.
  • the chip can then be inserted into a device as described above. The procedure is then carried out automatically. Carrying out an analysis of suspended particles takes approximately 1 to 20 minutes. During this time, approximately 10 to 20,000 cells flow through each microchannel. For the analysis of red blood cells, the amount of red blood cells from one drop of blood is sufficient.
  • the method is used in particular to analyze the shelf life of a blood supply.
  • the method can be used to check whether the blood reserve is still suitable for use by a patient.
  • patients can be protected from unsuitable blood supplies and on the other hand Blood supplies are protected from being destroyed if they have already been stored beyond their nominal shelf life.
  • the procedure can also be used to detect various abnormalities in red blood cells.
  • the effectiveness of relevant medications can be simulated or tested in vitro under physiological conditions.
  • novel drugs can be tested for sickle cell anemia.
  • the flow dynamics of the suspended particles are analyzed depending on the set pressure.
  • the resulting flow velocity can be calculated by identifying a suspended particle in at least two recording frames recorded at a defined time interval and determining the distance traveled between the two recording times. Based on the distorted speed of red blood cells and their deformability and characteristic shape, conclusions about diseases can be drawn, for example.
  • the shape of individual suspended particles is additionally or alternatively analyzed.
  • the shape of red blood cells - especially depending on the flow speed - provides an indication of possible diseases or also allows conclusions to be drawn about the shelf life of a blood supply.
  • the center of gravity of individual suspended particles is analyzed in relation to a microchannel center. From this, conclusions can be drawn about abnormalities or durability of red blood cells.
  • the analysis of the speed, shape and/or center of gravity of the particles is carried out in particular using automatic image recognition.
  • a machine learning method is provided for this purpose, which is trained to determine the shape of the suspended particles.
  • the location of the center of gravity of individual suspended particles and/or their speed can also be determined using classical methods or artificial intelligence.
  • the captured images are transmitted to a higher-level unit, where they are evaluated.
  • the report can, for example, show whether the blood cells have any abnormalities that indicate certain diseases or whether a blood reserve still meets the quality requirements to be used for a patient.
  • FIG. 1 shows a chip in a first embodiment of a perspective view obliquely from above
  • FIG. 3 shows a detail from FIG. 2 in an enlarged view, including the inlet and outlet area with filter elements
  • Fig. 4 shows the chip from Fig. 1 in a cross section along line IV-IV from Fig.
  • FIG. 5 shows the chip from FIG. 1 in a cross section along line V-V from FIG. 1 in a schematic representation
  • FIG. 6 shows the chip in a second embodiment in a cross section analogous to line IV-IV from FIG. 1 in a schematic representation
  • FIG. 7 shows the chip in a second embodiment in a cross section analogous to line V-V from FIG. 1 in a schematic representation
  • Fig. 8 shows a device for analyzing suspended particles in a perspective view obliquely from the front and Fig. 9 shows a detail of the device from Fig. 7 in a side view.
  • the chip 10 comprises a base body 12.
  • the base body 12 is designed in two parts and includes a channel element 14 and a carrier element 16 (clearly visible in FIGS. 4 and 5).
  • the chip 10 has a cover element 18.
  • the base body 12 - and here the channel element 14 - have several parallel microchannels 20 on a first surface (here on the underside) (cf. Fig. 3). In Fig. 5, only one microchannel 20 is shown as an example.
  • the microchannels 20 are designed as depressions in the base body 12 and are surrounded by the base body 12 on three sides. From the fourth side, the microchannels 20 are covered by the cover element 18 (see FIG. 5).
  • the channel longitudinal direction is the x direction
  • the channel transverse direction is the y direction
  • the channel vertical direction is the z direction.
  • the base body 12 is made of plastic, with the channel element 14 made of silicone and the carrier element 16 made of polycarbonate.
  • the cover element 18 is made of glass.
  • the base body 12 has a first reservoir 22 and a second reservoir 24 for receiving the suspended particles.
  • the first reservoir 22 and the second reservoir 24 are each a hollow cylinder which protrudes upwards from the base body 12.
  • the first reservoir 22 and the second reservoir 24 are connected to the plurality of microchannels 20 in a flow-conducting manner.
  • a molding part 82 is shown in FIG. Shapes are formed on this, which are formed on the base body 12 by molding. As shown in Fig.
  • an inlet 26 is formed on the base body 12 by molding the structures 26' on the side of the microchannels 20, which is connected to the first reservoir 22. It is also done by molding Forms 28 'forms an outlet which is connected to the second reservoir 24.
  • An inlet region 30 extends between the inlet 26 and the microchannels 20 and between the outlet and the microchannels 20, in which several filter elements can be arranged.
  • the inlet area 30 is formed by molds 30' and the filter elements by molds 32'.
  • the shapes 32' for the filter elements are cylindrical structures 32' which extend over the channel height in the z direction and which produce corresponding filter elements which prevent larger contaminants from getting into the microchannels 20.
  • the inlet area 30 is each designed in the shape of a drop and has a larger extent in the area of the inlet 26 or outlet, which decreases up to the microchannels 20. This is because the connection between the reservoir 22, 24 and the respective inlet 26 is subject to manufacturing tolerances and it should be ensured that a flow-conducting connection is established between the reservoir 22, 24 and the microchannels.
  • the chip 10 is designed to be mirror-symmetrical with respect to a y-z plane perpendicular to the length of the microchannels 20.
  • the chip 10 can thus be inserted into a device 48 in both directions (see FIGS. 8 and 9).
  • the suspended particles can be filled into one of the two reservoirs 22, 24 and flow through the microchannels 20 to the other reservoir 24, 22. There is no preferred direction of flow.
  • the base body 12 has a circumferential collar 34 (see FIGS. 1, 3 and 4).
  • the circumferential collar 34 is formed on the carrier element 16 in the first embodiment shown in FIGS. 1 to 5.
  • the connection between the carrier element 16 and the channel element 14 can be clearly seen in FIGS. 4 and 5.
  • the carrier element 16 has a plurality of openings 36, which are filled and engaged behind by connecting sections 37 of the channel element 14.
  • the connecting sections 37 form a smooth surface with the sections 39 that engage behind them.
  • the positive connection is achieved by the carrier element 16 already being present as a finished part during the production of the base body 12 and on the not yet hardened channel element 14 made of silicone. The silicone then passes through the openings 36 and engages behind them.
  • the chip 10 has a viewing window 38 (see FIGS. 1 and 5).
  • the viewing window 38 is an area of the base body 12 with reduced material thickness.
  • the base body 12 has a recess 40 here.
  • both the channel element 14 and the carrier element 16 have a smaller thickness than in the surrounding areas.
  • Fig. 5 it can be seen that an optical element 42 is injected in the area of the viewing window 38 between the carrier element 16 and the channel element 14.
  • the recess 40 for forming the viewing window 38 is surrounded by side walls 44 of the carrier element 16 and supported by it.
  • Ribs 41 extend between the viewing window 38 and the first reservoir 22, the second reservoir 24 and the circumferential collar 34 to stiffen the chip 10.
  • a riser 46 projects into the reservoir 22 and ends at a height in the reservoir 22 in which there is a desired concentration of sedimented particles.
  • FIG. 4 also already shows a part 66 of the device 48, which will be explained below in connection with FIGS. 8 and 9.
  • the first reservoir 22, the second reservoir 22, the riser 46, the circumferential collar 34 and the side walls 44 of the recess 40 for the viewing window 38 are formed by the carrier element 16.
  • the channel element 14 has a plurality of microchannels 20, the inlet 26, the outlet, the respective inlet area 30 and the filter elements 32 on its underside.
  • the base body 12 being formed in one piece here. That is, the first reservoir 22, the second reservoir 24, the riser 46, the circumferential collar 34, the viewing window 38, the microchannels 20, the inlet 26, the outlet, the respective inlet areas 30 and the filter elements 32 are all on the one-piece Base body 12 formed.
  • the base body 12 is made of TPE and made by plastic injection molding. It is also conceivable to produce the base body 12 from COC by injection molding.
  • the cover element 18 is also made of COC and has a material thickness of 140 pm. The connection between the base body 12 and the cover element 18 takes place here by means of diffusion bonding.
  • a device 48 is shown in FIGS. 8 and 9.
  • the device 48 is designed here as a compact table-top device and includes a housing 50.
  • the housing 50 is shown in FIGS. 8 and 9 without left and right side walls.
  • the housing 50 has a free space 52 in which an object stage 54 is accessible.
  • the object stage 54 is intended for positioning the chip 10.
  • the chip 10 is arranged in a recess (not visible) in the object stage 54.
  • the recess has a circumferential contact edge on which the circumferential collar 34 of the base body 12 comes to rest.
  • the top of the chip 10 is flush with the top of the object stage 54.
  • the optical unit 56 is arranged in the housing 50.
  • the optical unit 56 here comprises a lens 58 arranged below the chip 10 and a camera 60. This is an inverse arrangement. The light is redirected from the lens 58 to the camera 60 using suitable optics.
  • a light source 62 is arranged above the stage 54 and above the chip 10.
  • the light source 62 shines onto the chip 10 from the side facing away from the microchannels 20.
  • the optical unit 56 is on the side of the microchannels 20 with the lens 58 arranged. Overall, the device is designed to be operated in a transmitted light mode or brightfield mode.
  • a conveying device 64 is arranged in the housing 50 of the device 48, here a pump, which is connected to a connecting element 66 in a flow-conducting manner.
  • the connection element 66 projects into the free space 52 and serves to contact the first reservoir 22 of the chip 10.
  • the connection element 66 is designed here as a socket and also has a sealing ring 68 (see FIG. 4), so that the connection element 66 and that first reservoir 22 can be sealingly connected to one another.
  • the connection element 66 is connected to a spring 70, so that the connection element 66 can be pressed onto the chip 10 with a defined spring force.
  • the first reservoir 22 is pressurized via the connection element 66.
  • the pressure causes the suspended particles to flow from the first reservoir 22 through the microchannels 20 to the second reservoir 24.
  • the conveying device is connected here to a pressure accumulator.
  • a proportional valve (not visible) is arranged downstream of the conveying device 64, via which the pressure with which the first reservoir 22 is acted upon can be regulated. Depending on the pressure applied, the flowing suspended particles have a different speed, shape and position in relation to a center of a microchannel 20.
  • a gas connection 72 is also arranged downstream of the conveying device 64, via which the introduction of N2, O2 or CO2 or another process gas is possible.
  • the device 48 also has a heater.
  • the connection element 66 can be heated here in order to apply a desired temperature to the suspended particles.
  • the light source 62 and the connection element 66 are connected to a stage 74, which can be moved relative to the object stage 54 and the chip 10. After placing the chip 10 on the stage 54, the stage 74 is directed of the chip 10 until the connection element 66 is connected to the reservoir 22 with sufficient force. The light source 62 is then located close above the viewing window 38. After connecting the connection element 62 and the reservoir 22, the suspended particles can be analyzed.
  • the device has a cover 76, which can be lowered and closes the free space against accidental intervention. To start the analysis, a corresponding operating unit 80 is arranged on the housing 50.
  • the optical unit 56 is positioned relative to the chip 10 so that a field of view lies within the viewing window 38 of the base body 12.
  • the field of view and the microchannels 20 are dimensioned such that at least one microchannel 20 is always in the field of view of the optical unit 56 and the particles flowing therein can be analyzed.

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  • Health & Medical Sciences (AREA)
  • Dispersion Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
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  • Chemical Kinetics & Catalysis (AREA)
  • Hematology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Sampling And Sample Adjustment (AREA)

Abstract

L'invention concerne une puce (10) pour l'analyse de particules en suspension, la puce (10) comprenant un corps de base (12), ce corps de base (12) comportant au moins un microcanal (20) pour le passage des particules en suspension. Cette invention concerne en outre un dispositif (48), un système (78) et un procédé d'analyse de particules en suspension.
PCT/EP2023/058145 2022-03-30 2023-03-29 Puce, dispositif et procédé d'analyse de particules en suspension WO2023186984A2 (fr)

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DE102022107557 2022-03-30
DE102022107557.5 2022-03-30
DE102022107729.2 2022-03-31
DE102022107729.2A DE102022107729A1 (de) 2022-03-30 2022-03-31 Chip, Vorrichtung und Verfahren zur Analyse suspendierter Partikel

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WO2023186984A3 WO2023186984A3 (fr) 2023-11-23

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US8828226B2 (en) * 2003-03-01 2014-09-09 The Trustees Of Boston University System for assessing the efficacy of stored red blood cells using microvascular networks
KR20030061746A (ko) * 2003-06-23 2003-07-22 신세현 혈구 유변계
EP1687626A4 (fr) * 2003-06-23 2009-11-25 Sewon Meditech Inc Appareil pour mesurer la capacite de deformation des cellules sanguines
US10207266B2 (en) * 2015-09-29 2019-02-19 Foxconn Interconnect Technology Limited Microfluidic device for detecting cells of blood
JP2022521982A (ja) * 2019-02-26 2022-04-13 トルビアン サイエンシズ インコーポレイテッド アッセイ装置およびその使用方法
CA3156444A1 (fr) * 2019-10-30 2021-05-06 Umut Gurkan Biopuce ayant un microcanal pourvu d'un agent de capture pour effectuer une analyse cytologique
CN113433307A (zh) * 2020-02-10 2021-09-24 安派科生物医学科技有限公司 用于疾病检测和治疗的新型仪器和方法

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Title
DER VERÖFFENTLICHUNG KIHM AKAESTNER LWAGNER CQUINT S: "Classification of red blood cell shapes in flow using outlier tolerant machine learning", PLOS COMPUT BIOL, vol. 14, no. 6, 15 June 2018 (2018-06-15), pages el006278

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