WO2023186984A2

WO2023186984A2 - Chip, device and method for analysing suspended particles

Info

Publication number: WO2023186984A2
Application number: PCT/EP2023/058145
Authority: WO
Inventors: Stephan Quint
Original assignee: Cysmic GmbH
Priority date: 2022-03-30
Filing date: 2023-03-29
Publication date: 2023-10-05
Also published as: WO2023186984A3

Abstract

The invention relates to a chip (10) for the analysis of suspended particles, wherein the chip (10) comprises a base body (12), wherein the base body (12) has at least one microchannel (20) for the passage of the suspended particles. The invention also relates to a device (48), a system (78) and a method for analysing suspended particles.

Description

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. In particular, 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.

Specifically, 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 dynamics of flowing particles such as microbeads, cells, microbes, protozoa and vesicles under microfluidic conditions is the subject of many research projects, which result in a whole range of possible applications. In relevant studies, the interaction between the flow and the particles is examined Experimental approaches and numerical models are being developed to describe the interaction dynamics in the micrometer or even submicrometer range and to improve their understanding.

An important question is the influence of external forces on the particles, which are, for example, forces from the surrounding fluid (local or global pressure or flow changes), light-induced forces (e.g. optical tweezers), or forces due to static or dynamic electrical and /or magnetic fields can act. If such an external force acts on a particle, the shape of non-rigid particles is usually influenced. The The resulting shape is often characteristic of the individual particle type or subtype.

So is e.g. B. in the flow of red blood cells in microcapillary blood vessels, their shape is of great importance for the flow behavior and thus their transport, e.g. their speed. For a given mechanical constriction of a certain size and cross-section, at relatively low flow velocities, the so-called "parachute" shape of the cells is induced, which has a large cross-sectional area. It ensures a sufficient transport speed of the red blood cells to enable sufficient gas exchange with the surrounding tissue. At relatively high flow velocities, however, the red blood cells tend to take on the so-called asymmetrical “slipper” shape. This reduces the relative transport speed compared to the surrounding medium, as shown in the publication Kihm A, Kaestner L, Wagner C, Quint S, Classification of red blood cell shapes in flow using outlier tolerant machine learning, PLoS Com put Biol. 2018 Jun 15; 14(6):el006278. dot 10.1371/journal.pcbl.1006278. PMID: 29906283; PMCID: PMC6021115 is known. This feedback mechanism, based on the external shape of individual cells, is a small but important part of a higher-level control mechanism that regulates the gas exchange (oxygen/carbon dioxide) of the entire organism. If the flow speed is varied, a different cell shape predominates for each speed, and a transition from "parachutes" to "slippers" can be observed. The underlying statistical distribution is called a “phase diagram.”

Studies have shown that this cell-based feedback mechanism can be disrupted by inherited or acquired diseases, parasites (malaria) or even medications. It was also found that the age of cells and the storage time, e.g. of blood transfusions, can influence this mechanism.

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. Furthermore, 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. In particular, the at least one microchannel is rectangular, semicircular or round in cross section or trapezoidal or rectangular with round corners. For the analysis of red blood cells, 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. As already described in the introduction, such 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.

In a practical embodiment of the chip according to the invention, the base body has several, in particular parallel (or in a relative Angles of +/- 10 degrees to each other), micro channels. In particular, 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. In this respect, an xy adjustable table can be dispensed with when analyzing particles flowing through the chip using a device mentioned above. In particular, 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. Analogously, 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. Alternatively, 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.

If the chip comprises a cover element made of glass or plastic, 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.

As an alternative to the above-described environment of the at least one microchannel of the base body and the cover element, the at least one microchannel can also be designed completely as a cavity in the base body.

In particular, 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. In particular, 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.

In particular, 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. With suspended red blood cells, it is common for sediment to settle at the bottom of the suspension container after a certain storage period. In order to prevent the lowest layers of sediment and As a result, increased cell concentrations are passed through the at least one microchannel, the riser pipe branches off in the suspension container at a distance from the bottom of the reservoir. The height of the riser in the reservoir is selected so that the density of suspended particles is as constant as possible even under sedimentation and the lowest sediment layers are not included. The diameter of the riser is also chosen so that the sediment layers are not stirred up.

Alternatively, it is also conceivable that 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.

In a further practical embodiment of the chip according to the invention, 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. In particular, a filter is arranged at both ends of a microchannel. In this case, 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. For example, 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.

In particular, 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. For example, 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.

In a further practical embodiment of the chip, 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.

In a further practical embodiment of the chip according to the invention, 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. In particular, 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. To form the viewing window, 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.

For viewing windows, 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.

Furthermore, the base body can have at least one positioning aid. The base body preferably has several positioning aids distributed around the viewing window. For example, 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. For example, 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.

In a further practical embodiment of the chip according to the invention, the base body has an at least partially circumferential collar. In particular, 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. 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.

In particular, 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.

In particular, 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.

In a practical embodiment of the method according to the invention, the cover element and the base body are subjected to a plasma treatment before they are connected. In particular, it 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. After the plasma treatment of the surfaces of the base body and the cover element intended for the connection, these surfaces are brought into connection and form stable connections. 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. In addition, the connection can be established reproducibly and with a reasonable amount of time.

Alternatively, the cover element and the base body are connected using UV bonding, solvent bonding, diffusion bonding or adhesively (see above).

It can also be provided that 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. In step e), the connected elements of the carrier element and channel element are then removed.

Preferably, in the method described above for producing the two-part base body in step c), 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.

In particular, 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.

As already explained above, 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. In the area of the viewing window, an optical element can also be injected into the base body.

As an alternative to the process described above, 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. For analyzing individual red blood cells, 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. Furthermore, 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.

The 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. For the sealing connection of the connection element and the chip (here in particular with the reservoir), 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. As already described above, 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. In particular, the at least one microchannel is not damaged. This is particularly advantageous for chips that comprise a comparatively soft material such as silicone.

In order to create conditions that are as realistic as possible for the analysis of the suspended particles, in particular red blood cells, the device in particular has a heating device. With 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. In particular, 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.

In a further practical embodiment of the device, 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. Alternatively, a white light source or a light source that emits light in the red wavelength range (640 nm - 780 nm) 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. In this case, 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. In this configuration, 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.

The object stage and the at least one connection element can in particular be moved relative to one another. After positioning the chip, the 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.

In a further practical embodiment of the device according to the invention, it has a conveyor device. In particular, 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. In particular, the conveying device is connected to a pressure accumulator. For example, 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.

Furthermore, 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. In particular, 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.

As already indicated above, after images have been captured by the optical unit, an image evaluation takes place. This is carried out locally or in a higher-level unit - in the cloud. In particular, 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. In particular, 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. In particular, 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. In particular, 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. For this purpose, 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).

In a practical embodiment of the system, it has a gas connection for supplying filtered room air, N2, CO2, O2 and/or other process gases into the suspension. In particular, through oxygenation or deoxygenation it is possible to change the properties of the suspended particles. This offers another possibility to analyze the particles and, for example, to check suspended red blood cells for abnormalities. In particular, the suspended particles are suspended in a buffer solution. In the case of red blood cells, 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. However, it is also conceivable to measure the red blood cells directly in plasma or another physiological buffer. In particular, 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.

To carry out the method, 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. For the analysis of red blood cells, 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. In particular, the method can be used to check whether the blood reserve is still suitable for use by a patient. On the one hand, 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. In addition, the effectiveness of relevant medications can be simulated or tested in vitro under physiological conditions. For example, novel drugs can be tested for sickle cell anemia.

In particular, 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.

In particular, 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.

Alternatively or in addition to the two above properties of the particles, 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. In particular, 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.

After the evaluation has been carried out, a report with the analysis results is created. In the case of the analysis of red blood cells, 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.

Further practical embodiments and advantages are described below in connection with the figures. Show it:

1 shows a chip in a first embodiment of a perspective view obliquely from above,

2 shows the impression part for forming the microchannels,

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.

1 in a schematic representation,

5 shows the chip from FIG. 1 in a cross section along line V-V from FIG. 1 in a schematic representation,

6 shows the chip in a second embodiment in a cross section analogous to line IV-IV from FIG. 1 in a schematic representation,

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.

1 shows a chip 1 for analyzing suspended particles in a first embodiment. The chip 10 comprises a base body 12. In the first embodiment shown here, 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). Furthermore, 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).

Here, the channel longitudinal direction is the x direction, the channel transverse direction is the y direction and the channel vertical direction is the z direction.

In the first embodiment of the chip 10, 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. In the present case, 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.

2, 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.

As can be clearly seen in FIGS. 1 and 2, 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.

Furthermore, 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.

Furthermore, 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. In the first embodiment of the chip 10, both the channel element 14 and the carrier element 16 have a smaller thickness than in the surrounding areas. In 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.

4 shows the transition from the first reservoir 22 to the inlet 26 into the microchannels 20. 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.

4 also already shows a part 66 of the device 48, which will be explained below in connection with FIGS. 8 and 9.

According to the first embodiment, 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.

In the following, the same reference numbers are used to describe further embodiments for identical or at least functionally identical components as for the description of the first embodiment. 6 and 7 show a second embodiment of the chip 10 according to the invention, 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. In this case, 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, together with the chip 10, forms a system 78 for analyzing suspended particles.

The device 48 is designed here as a compact table-top device and includes a housing 50. In order to be able to see the interior of the device 48, 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.

An 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.

Reference symbol list

10 chips

12 basic bodies

14 channel element

16 carrier element

18 cover element

20 microchannel

20' projection for molding the microchannel

22 first reservoir

24 second reservoir

26 entrance

26' mold for molding an inlet

28' mold for molding an outlet

30 entrance area

30' mold for molding an inlet area

32' mold for molding a filter element

34 collars

36 opening

37 connecting section

38 viewing windows

39 rear section

40 deepening

41 rib

42 mask

44 sidewall

46 riser

48 device

50 cases

52 open space

54 object table

56 optical unit

58 lens 60 camera

62 light source

64 conveyor/pump

66 connection element 68 sealing ring

70 feather

72 gas connection

74 stage

76 Cover 78 System

80 control element

82 impression part

Claims

Claims Chip for the analysis of suspended particles, the chip (10) comprising a base body (12), the base body (12) having at least one microchannel (20) for the passage of the suspended particles. Chip according to the preceding claim, characterized in that the base body (12) has a plurality of microchannels (20). Chip according to one of the preceding claims, characterized in that the base body (12) is made of glass, ceramic or plastic. Chip according to one of the preceding claims, characterized in that the at least one microchannel (20) is covered by a cover element (18). Chip according to the preceding claim, characterized in that the cover element (18) is made of glass or plastic. Chip according to one of the preceding claims, characterized in that the base body (12) has at least one reservoir (22, 24) for storing the suspended particles. Chip according to the preceding claim, characterized in that in the at least one reservoir (22, 24) a riser (46) ends, which establishes a flow-conducting connection between the reservoir (22, 24) and the at least one microchannel (20), whereby the riser (46) ends above the bottom of the reservoir (22, 24) and/or a funnel-shaped opening is formed in the bottom of the reservoir (22, 24). Chip according to one of the preceding claims, characterized in that at least one filter (26) is arranged at at least one end of the at least one microchannel (20). Chip according to one of the preceding claims, characterized in that the chip (10) is mirror-symmetrical with respect to a yz plane perpendicular to the at least one microchannel (20). Chip according to one of the preceding claims, characterized in that at least one microchannel (20) has a narrowing. Chip according to one of the preceding claims, characterized in that the base body (12) is formed at least in two parts and comprises a channel element (14) and a carrier element (16), the channel element (14) has at least one microchannel (20) and wherein the carrier element (16) is arranged on the channel element (14) on a surface facing away from the at least one microchannel (20). Chip according to the preceding claim, characterized in that the carrier element (16) is made of glass, ceramic or plastic. Chip according to one of the two preceding claims, characterized in that the carrier element (16) and the channel element (14) are captively connected to one another. Chip according to the preceding claim, characterized in that at least one connecting section (37) is formed on the surface of the channel element (14) facing the carrier element (18), wherein the connecting section (37) extends through a corresponding opening (36) in the carrier element (16) and engages behind it. Chip according to one of the preceding claims, characterized in that the base body (12) has a viewing window (38). Chip according to one of the preceding claims, characterized in that the base body (12) has at least one positioning aid. Chip according to one of the preceding claims, characterized in that the base body (12) has an at least partially circumferential collar (34). Use of a chip (10) according to one of the preceding claims for analyzing suspended particles. Method for producing a chip (10) for the analysis of suspended particles, comprising the following method steps a) providing a structured impression part (82) with at least one elevation (20') for structuring a microchannel (20), b) inserting the impression part (82) into a mold, c) filling material into the mold to form a base body (12), d) hardening the base body (12), e) removing the base body (12) molded from the molding part (82) from the mold, f) arranging a cover element (18) on a first surface of the base body (12), on which the at least one microchannel (20) is formed. Method according to the preceding claim, characterized in that the base body (12) is produced in step c) from plastic by injection molding. experienced according to one of the two preceding claims, characterized in that the cover element (18) and the base body (12) are subjected to a plasma, UV and/or solvent treatment before they are connected. Method according to one of the three preceding claims, characterized in that the base body (12) is manufactured in two parts and in step c) a material is filled to form a channel element (14) and then a carrier element (16) is placed on the channel element (12). is, and in step e) the elements support element (16) and channel element (14) are removed. Method according to the preceding claim, characterized in that the carrier element (16) in step c) is already present as a hardened injection-molded part made of plastic. Method according to one of the two preceding claims, characterized in that the support element (16) is placed on the channel element (14) with such a pressure that the material of the channel element (14) comes out of at least one opening (36) in the support element ( 16) emerges and engages behind this opening (36). Method according to one of the preceding claims, characterized in that a flow-conducting connection from a reservoir (22, 24) to the at least one microchannel (20) is produced by piercing the base body (12). Method for producing a chip (10) according to one of the preceding claims 1 to 17 by means of additive manufacturing. Device for analyzing suspended particles, with an optical unit (56) and with an object stage (54) for positioning a chip (10) with at least one microchannel (20) for the flow of the suspended particles, and with a connection element (66) for producing a flow-conducting connection to the at least one chip (10). Device according to the preceding claim, characterized in that the connecting element (66) can be pressed onto the chip (10) by means of spring force. Device according to one of the preceding claims, characterized in that the object table (54) has a recess with an at least partially circumferential contact edge, against which a corresponding collar (34) of the chip (10) can be brought into contact. Device according to one of the preceding claims, characterized in that the device (48) has a heating device. Device according to one of the preceding claims, characterized in that the optical unit (56) is pivotably mounted. Device according to one of the preceding claims, characterized in that it is designed to work in a transmitted light mode. Device according to one of the preceding claims, characterized in that the object stage (54) and the at least one connection element (66) can be moved relative to one another. Device according to one of the preceding claims, characterized in that it has a conveyor device (64). Device according to one of the preceding claims, characterized in that this is set up to analyze the position of the center of gravity of individual suspended particles relative to a center of a microchannel (20). Device according to one of the preceding claims, characterized in that it has a communication device for communication with a higher-level unit. Use of a device (48) according to one of the preceding claims for analyzing suspended particles. System for analyzing suspended particles, with a device (48) with an optical unit (56) and an object stage (54), a chip (10) with at least one microchannel (20) being arranged on the object table (54), and with a conveying device (64) for conveying the suspended particles from a first reservoir (22) through the at least one microchannel (20) to a second reservoir (24) and wherein by means of the optical unit (56) the suspended particles in the at least one Microchannel (20) can be viewed. System according to the preceding claim, characterized in that the pressure generated by the conveying device (64) on the suspended particles in the at least one microchannel (20) can be changed by a proportional valve. System according to one of the preceding claims, characterized in that a gas connection (72) is provided for supplying filtered room air, N2, CO2, O2 or another process gas to the suspended particles. Method for analyzing suspended particles, wherein the particles are passed through a chip (10) with at least one microchannel (20) and the particles flowing through the at least one microchannel (20). are recorded with a camera (60) and the suspended particles are analyzed using image evaluation. experienced according to the preceding claim, characterized in that the flow velocity of the suspended particles is analyzed. experienced according to one of the two preceding claims, characterized in that the shape of individual suspended particles is analyzed. Method according to one of the three preceding claims, characterized in that the center of gravity of individual suspended particles is analyzed in relation to a center of a microchannel (20). Method according to one of the preceding claims, characterized in that the analysis is carried out using automatic image recognition. Method according to one of the preceding claims, characterized in that the recorded images are transmitted to a higher-level unit. Method according to one of the preceding claims, characterized in that after the evaluation, a report with the analysis results is created.