Fluidic structure and method for low-loss and gentle storage and transport of objects in a dispersion

Abstract

The invention relates to a fluidic structure with a reservoir (14) and a channel (16) and a method for low-loss and gentle storage and transport of objects in a dispersion.The method comprises the following steps: providing a fluidic structure, introducing a mixture of a carrier fluid, a shear-thinning medium and the objects dispersed therein into the reservoir (14), transporting at least a part of the mixture from the reservoir (14) through the channel (16) at a flow rate wherein the mixing ratio of the carrier fluid and the shear-thinning medium on the one hand and the flow rate in the fluidic structure on the other hand are set such that the mixture at rest or at lower transport speeds has a sufficiently high viscosity to prevent the objects from settling, and that at higher transport speeds the objects in the mixture are moved from the channel wall to the center of the channel and transported further there due to the shear-thinning behavior of the mixture.

Description

[0001] The invention relates to a fluidic structure and a method for the low-loss and gentle storage and transport of objects in a dispersion within a microfluidic system, such as a microfluidic chip or a microfluidic cartridge. The invention relates to the field of microfluidics, where samples, in this case a dispersion, are processed in volume ranges from 1 µl to 10 ml.

[0002] Microfluidics has long been the subject of biotechnological research and development and is used, for example, in the form of so-called lab-on-a-chips, including for medical diagnostics in point-of-care products. On these microfluidic chips, protocols previously developed in the laboratory are implemented as completely as possible in a microfluidic structure on the lab-on-a-chip, allowing the protocols to run largely automatically with minimal manual intervention. The chips are typically used with operator devices, which are equipped with a slot for the chip and, if necessary, electrical, fluidic, and actuator interfaces to the chip.

[0003] These and other microfluidic systems contain various fluidic structures with dimensions in the millimeter or micrometer range. Microfluidic systems often feature a base plate (substrate) with grooves and depressions formed within it, and a cover film or lid that seals these grooves and depressions and is laminated onto the substrate. The base plates are molded from plastic using injection molding or embossing processes, and the cover films are bonded to the base plates in a fluid-tight manner using adhesive or welding processes.

[0004] Dispersions (suspensions or emulsions) are known to tend towards sedimentation or segregation of the objects dispersed in the liquid (particles, cells, droplets, etc.). In fluidic structures, and especially in microfluidic structures with capillary dimensions of, for example, 100 µm or less, very short sedimentation distances can occur relative to gravity, depending on the orientation of the transport and storage structures. This allows the objects to sediment and adhere to surfaces within short timeframes (seconds or less). This problem arises in all applications where objects are stored in dispersions or transported in fluidic systems, particularly with low flow rates, and where sedimentation and thus segregation of the objects can occur during this application.

[0005] Microfluidic flow cytometry serves as an example. Before analysis in the measuring channel, the cell suspension must be stored in a reservoir. During the analysis, cells can sediment, primarily due to density differences, and subsequently even adhere to the channel or chamber walls. The flow rate during emptying the reservoir is then often insufficient to transport all the cells through the measuring section. This results in errors in the count rates. Furthermore, typical biological cell suspensions contain different cell types that vary in size, density, shape, and / or other physical properties, and therefore sediment at different rates. Thus, relative count rates are also distorted.

[0006] These effects become increasingly important as sample volumes decrease. Particularly in microfluidic systems, sedimentation and subsequent adhesion of objects can lead to significant losses, since sedimentation distances are shorter and, due to the large surface area to volume ratio, a proportionally larger surface area is available for adhesion. Furthermore, sedimentation and adhesion of objects, especially in microfluidic systems, can lead to clogging of the microchannels.

[0007] Sedimentation or segregation also has a particularly negative impact on applications that require hydrodynamic focusing to transport the sample stream in a defined (optical) plane. Even low sedimentation rates cause the objects in such applications to leave the focused sample stream and become undetectable.

[0008] At the applicant's facility, countermeasures to prevent sedimentation or segregation have so far included minimizing storage time, minimizing measurement time, increasing flow rates, aligning storage vessels, using rinsing solutions, adjusting the medium's density to the object density, and using shear-thinning media for storing cell suspensions immediately before analysis, provided the respective application fundamentally permitted one or more of these countermeasures. These compensatory measures, however, in turn impair the processes occurring within the fluidic structure. For example, if high flow rates are used to compensate for sedimentation, very high pressures quickly arise in the (micro)fluidic systems, which individual components cannot withstand. The effects on transported biological objects such as cells are equally serious.The higher the flow rates, the more the cell is sheared in the flow and potentially destroyed. This makes non-destructive analysis more difficult.

[0009] The object of the present invention is therefore to provide a fluidic structure and a method for the low-loss and gentle transport of objects in a dispersion, which, particularly in microfluidic systems, avoids or at least significantly reduces sedimentation or separation of objects in a dispersion as universally, cost-effectively and reliably as possible.

[0010] The problem is solved by a fluidic structure having the features of claim 1 and a method having the features of claim 11.

[0011] The fluidic structure for the low-loss and gentle storage and transport of objects in a dispersion comprises a reservoir and a channel for the dispersion. The reservoir has a filling opening and an outlet opening to the channel, with a transport direction defined perpendicular to the outlet opening. The reservoir has a cross-sectional area perpendicular to the transport direction. The reservoir further comprises at least one tapered section in which the cross-sectional area decreases along the transport direction from a maximum value F max to a minimum value F min < F max funnel-shaped tapering and where the minimum value F min corresponds to the cross-sectional area of ​​the mouth opening.

[0012] The channel geometry according to the invention ensures, due to the maximum cross-sectional area at the reservoir inlet, that the reservoir can be filled easily. The cross-sectional area of ​​the channel F Kperpendicular to the transport direction is preferably equal to the cross-sectional area of ​​the mouth opening, i.e., preferably equal to F min , and in any case smaller than the maximum cross-sectional area F max Due to the funnel-shaped narrowing from the maximum cross-section to the channel cross-section, a continuous transition is provided, during which the flow velocity constantly increases in the transport direction. This alters the shear forces and wall effects acting in the fluid in a controlled manner, which, in conjunction with a correspondingly adapted shear-thinning behavior of the fluid, effectively prevents sedimentation both during static storage of the dispersion in the fluidic structure and during dynamic transport.

[0013] Preferably, the ratio of the maximum value to the minimum value is 5 < F max / F min < 10 6 .

[0014] F maxlargely determines the volume of the reservoir, while F min The maximum flow velocity in the transition zone from the reservoir to the channel is determined. The lower limit is 5 < F max / F min The ratio is determined on the one hand by a practical minimum size of the reservoir, since otherwise the liquid can only be poured into the reservoir with great effort, and on the other hand by the fact that F min due to the generally small volumes to be treated, it cannot be chosen to be arbitrarily large.

[0015] The upper limit F max / F min < 10 6 is conversely, by the cross-sectional area of ​​the channel F KThe specified minimum dimensions are 10 µm × 10 µm. Such small channels are just about suitable for handling bacteria. Larger reservoirs are also impractical, as processing larger volumes at realistic pressures would take far too long.

[0016] The reservoir preferably has a volume V R and the channel has a volume V K on, where V is the ratio of the volume of the reservoir to the volume of the channel. K < V R applies and where the volume of the reservoir V R preferably in the range of 0.1 ml to 10.0 ml and particularly preferably in the range of 0.1 ml to 2.0 ml.

[0017] The fluidic structure is preferably formed in a plate-shaped substrate which defines a coordinate system in three mutually perpendicular directions with a depth direction d, a height direction h and a width direction w, wherein the reservoir is formed by a depression and the channel by a channel in the substrate and wherein the transport direction coincides with the height direction.

[0018] Such a substrate includes the aforementioned base plates with grooves and depressions formed within them, which are closed on their open side by means of a cover film laminated onto the substrate. The thickness d of the essentially flat, plate-shaped substrates limits the possible depth of the structures.

[0019] The cross-sectional area of ​​the reservoir in the region of the maximum value F maxIt therefore also has a minimum extent in the depth direction and a maximum extent in the width direction, with the aspect ratio between the maximum extent and the minimum extent of the cross-sectional area being in the range of 1 to 30, preferably in the range of 3 to 20.

[0020] Preferably, the cross-sectional area of ​​the reservoir decreases in the tapering section along the transport direction in the lateral direction.

[0021] The change in cross-section can be manufactured most accurately in this direction.

[0022] At least one first wall, which limits the reservoir in the width direction, defines a normal direction at each point of the first wall, which in the tapered section includes an angle α with the transport direction, for which 95° < α, preferably 100° < α, and 170° > α, preferably 165° > α.

[0023] The transport direction of the microfluidic system preferably coincides with the direction of gravity during intended use. Due to the upper limit of the angle α of 170°, preferably 165°, the normal on the first wall always has a component pointing in the lateral direction; that is, the first wall has no horizontal orientation at any point. The upper limit of the angle α of 170°, and preferably 165°, creates a sufficient inclination along this wall to reduce the risk of sedimentation.

[0024] On the other hand, the lower limit of 95°, preferably 100°, stipulates that the normal on the first wall always has an upward-pointing component, thus ensuring that the wall is not too steep and the overall taper is not too high. Otherwise, the first wall in the taper section can have any gradient, increasing or decreasing, within the limits of the surface normal.

[0025] The narrowing section itself can provide a significant portion of the reservoir's volume. Upstream of the narrowing section, the reservoir can also include a section with a constant or even decreasing cross-sectional area, but preferably with a constant cross-sectional area F. maxThe reservoir must have a tapered shape to increase the volume to the required size without increasing its width. In the case of a tapered section, the angle α is < 90°, which is not entirely impossible since sedimentation cannot occur on such surfaces. To maximize surface area utilization on the substrate, the first wall, which defines the reservoir's width, preferably runs parallel to the direction of transport in the section with a constant cross-sectional area. In both cases, the first wall has a kink at the transition between the tapered section and the section with a constant cross-sectional area.

[0026] A second wall, which limits the reservoir in the lateral direction, lies opposite the first wall. This second wall can optionally run parallel to the transport direction and thus not contribute to the narrowing, or it can also have a normal direction that forms an angle α > 90° with the transport direction in the narrowing section, thus also contributing to the narrowing.

[0027] Advantageously, the reservoir has a filling opening on a wall opposite the direction of transport of the outlet, wherein the outlet and the filling opening are arranged transversely to the direction of transport.

[0028] The outlet opening and the filling opening preferably do not overlap in a projection in the direction of transport.

[0029] A filling opening offset perpendicular to the direction of transport and thus perpendicular to the direction of gravity, and in particular without overlap, prevents the liquid from directly hitting the outlet opening when filling the reservoir and trapping air there, which would then only be possible to remove from the channel with considerable effort. A droplet of liquid introduced into the reservoir through the offset filling opening falls directly onto the first wall in the narrowing section and flows down this wall in a controlled manner to the outlet opening, while the air is gradually displaced from there.

[0030] Preferably, the channel includes a buffer channel for bubble-free filling of the fluidic structure, wherein the buffer channel further preferably has an interface for connecting a pump on its side opposite the outlet. This pump can be configured to both draw a fluid (buffer solution, dispersion) into or out of the buffer channel, as well as to push a fluid (buffer solution, dispersion) into or out of the buffer channel.

[0031] Furthermore, the buffer channel preferably has a cross-sectional area F P on, where the ratio from the maximum value F max and the cross-sectional area F P The following applies: 5 < F max / F P< 1000. In a preferred embodiment of the invention, the buffer channel has a meandering shape at least in sections in order to create a sufficiently large volume for a supply of the dispersion and at the same time to be arranged on the substrate in a space-saving manner.

[0032] In another preferred embodiment, the channel comprises a functional channel with a cross-sectional area F M , where the ratio is the maximum value F max and the cross-sectional area F M Preferably: 100 < F max / F M < 10 6 .

[0033] A "functional channel" is a channel section following the main channel in which the objects are subjected to process steps such as detection, analysis, singulation or dispensing, i.e., for example, a measuring channel, detection channel or analysis channel or the like.

[0034] Preferably the maximum value Fmax the cross-sectional area of ​​the reservoir of 10 mm 2 up to 50 mm 2 Preferably, the cross-sectional area of ​​the buffer channel is F. P of 0.1 mm 2 up to 5 mm 2 Preferably, the cross-sectional area of ​​the functional channel is F. M of 100 µm 2 up to 0.25 mm 2 In contrast to the buffer channel, the functional channel is generally narrower. The buffer channel typically has a cross-sectional area with a low aspect ratio of <2 to provide maximum volume in a small space. In contrast, the functional channel has an aspect ratio adapted to its specific function. For example, in the case of a measurement channel, this might be greater than 5.

[0035] In a further preferred embodiment of the invention, the channel comprises a buffer channel and a functional channel fluidically connected to the buffer channel. Funnel-shaped tapered cross-sectional transitions can be arranged between them.

[0036] The fluidic structure advantageously includes one or more valves that selectively open or close sections of the channel. These valves allow the movement of the dispersion or other fluids within the fluidic structure to be controlled. For example, in a first step, the dispersion or fluid can be drawn from the reservoir into the buffer channel by means of a pump connected to the interface of the buffer channel, and then, in a second step, after switching the valve(s), it can be pumped from the buffer channel into the functional channel.

[0037] The aforementioned task is further solved by a method for low-loss and gentle storage and transport of objects in a dispersion, comprising the following steps: - Provide a fluidic structure as described above, - Introducing a mixture of a carrier fluid, a shear-thinning medium and the objects dispersed therein into the reservoir, - Transporting at least part of the mixture from the reservoir through the channel at a flow rate, - wherein the mixing ratio of the carrier fluid and the shear-thinning medium on the one hand and the flow rate in the fluidic structure on the other hand are set such that the mixture has a sufficiently high viscosity at rest or at lower transport speeds to prevent the objects from sedimenting, and that at higher transport speeds the objects in the mixture are moved from the channel wall to the center of the channel and transported further there due to the shear-thinning behavior of the mixture.

[0038] The order in which the carrier fluid is mixed with the shear-thinning medium and with the objects is not of primary importance to the invention. The method works regardless of whether the shear-thinning medium is added to the fluid first and then the objects are suspended in the fluid, or vice versa. The mixing is preferably carried out outside the fluidic structure, but can also, in principle, take place within the fluidic structure.

[0039] In this context, without limitation to specific applications, a dispersion is defined as any mixture of the carrier liquid with solid, undissolved particles or with finely dispersed droplets of a second liquid without visible separation or phase boundary. A dispersion thus includes both suspensions and emulsions. For the purposes of this document, the carrier liquid can be (except for tolerable impurities) a pure liquid, a liquid solution, or itself a dispersion.

[0040] The term "object" refers to all types of solids, including biological cells and microorganisms, as well as small volumes of another liquid completely enclosed by the liquid (dispersed phase, droplets).

[0041] Examples of the investigational dispersion are: - Blood: Suspension of blood cells (white, red, platelets) and other target objects suspended therein, such as CTCs, whereby blood per se is shear-thinning primarily due to the behavior of red blood cells; - Milk: Droplet emulsion with slightly shear-thinning properties. However, other target objects, such as bacteria, may be dissolved in the milk; - A homogeneous shear-viscous medium, such as a xanthan gum solution, which is used as a transport medium for the cells; - Dispersions in which cells are contained within droplets, which in turn are suspended in a shear-thinning medium. For example, to create different environments within the droplet for individual cells and then sort them based on their response to these environments.

[0042] The channel geometry and the set conveying or flow rate determine the transport velocities and thus the shear forces in the fluidic structure. Within the reservoir, the dispersion is either at rest or, during transport, moves at a typical transport velocity of 0 µm / s to 10 mm / s, relative to regions of the fluidic structure with cross-sectional area F. max In the buffer channel, the transport speed is typically between 10 µm / s and 25 mm / s. Higher transport speeds in the range of mm / s are used during filling, and lower transport speeds in the range of 10,000–15,000 µm / s are used during transport to the functional channel. Within the functional channel, the speed ranges from 1 mm / s to 1,000 mm / s.

[0043] The properties of shear-thinning media are already strongly pronounced at very low concentrations.

[0044] As soon as shear forces act on a shear-thinning medium, its viscosity decreases. Therefore, the pressure differential required to convey a shear-thinning medium decreases as the shear force increases. In a fluidic system, this means that the pressure drop is reduced, particularly in the often desirable case of high flow rates and small channel cross-sections. This makes it possible to pump shear-thinning media through small channel systems, such as those of a microfluidic chip, even at high flow rates, without risking damage to the channel structure or the bond between the cover and the substrate.

[0045] In Newtonian fluids, there are hydrodynamic forces that, on the one hand, displace objects away from the channel wall in the direction of higher flow velocity. These forces decrease with increasing distance from the wall. On the other hand, there is a precisely opposing force that displaces objects from the center of the channel, where the flow velocity is highest and the curvature of the river profile is greatest, towards the less curvature of the river profile. At a certain distance from the channel wall and the center of the channel (locations of highest flow velocity), these two forces are in equilibrium, and objects are transported at this distance from the wall (the "equilibrium position").

[0046] For shear-thinning media, another force comes into play, displacing objects from areas of high shear to areas of low shear. High shear is present at the channel wall, and the lowest shear is found in the center of the channel. For media with sufficiently strong shear-thinning properties, this additional force can cause objects to reach the center of the channel after traveling very short distances. There, they are transported at the position with the least curvature in the flow profile, where the lowest shear force acts upon them.

[0047] This results in at least three advantages. First, the adhesion of objects to the channel walls during transport and storage is efficiently prevented. To optimally utilize this effect, it is advantageous to establish high shear forces in the areas of the flow profile near the walls, i.e., to use flow rates and small channel dimensions in at least one cross-sectional direction. Even in more complex fluidic systems with bends and intersections, object adhesion can be efficiently prevented.

[0048] Secondly, due to the lower shear rates in the center of the channel, the objects are transported through the fluidic system as gently as possible. This allows for non-destructive analysis with minimal stress on the objects, especially biological objects such as cells or microorganisms. Furthermore, in many applications, such as flow cytometry, the objects must be focused in at least one plane. This is usually achieved mechanically by introducing enveloping flows according to the principle of hydrodynamic focusing. This effort is no longer necessary, since in the case of the method according to the invention, the objects are transported in the center solely by the non-Newtonian fluid, even without limiting elements. If enveloping flows are required for other processing purposes, for example,However, if further substances are to be added, this is still possible without affecting the described effect of focusing and sedimentation avoidance.

[0049] Thirdly, unlike the two transport positions on the flanks in a Newtonian medium, only one transport position exists in a non-Newtonian medium. This offers the advantage that no two objects are transported side by side in a channel. This ensures that all objects in a channel can always be detected sequentially, or that, for example, sorting can be carried out precisely. At the same time, the objects are transported in one plane or exactly on a channel center axis, so that they are located in the same object plane and facilitate optical detection, for example, when they lie in the focal plane of a (microscopic) objective or in the plane of maximum light-gathering efficiency of a photodetector.

[0050] The invention combines, for the first time, the properties of shear-thinning media with the fluidic structure geometry discussed above, such that the mixing ratio of the carrier fluid and the shear-thinning medium, on the one hand, and the flow rate, on the other, are adjusted to prevent the objects from settling and simultaneously transport them gently. This is achieved when the mixture, at rest or at lower transport speeds within large channel cross-sections, exhibits a sufficiently high viscosity to prevent the objects from settling, and when, at higher transport speeds within small channel cross-sections, the objects in the mixture are moved from the channel wall to the center of the channel and transported further there due to the shear-thinning media properties described above. Thus, sedimentation is prevented even in the moving fluid.The relative terms "lower transport speed" and "higher transport speed" refer only to each other; that is, the statement made here is simply that higher transport speed > lower transport speed. The absolute values ​​of both lie within the ranges specified above.

[0051] A crucial parameter here is the shear rate contrast, which expresses the change in viscosity as a function of the shear rate. Sufficient shear rate contrast ensures that the particles concentrate in the center of the channel and are transported there. Simultaneously, it prevents sedimentation due to high viscosity in stationary fluids. Finally, it prevents high pressure losses, allowing the implementation in a microfluidic structure to be achieved with relatively simple means. A viscosity difference of 0.1 s is particularly preferred over a shear rate interval of 0.1 s. -1 up to 1000 s -1 at least two orders of magnitude.

[0052] In particular, the concentration of the shear-thinning medium in the mixture is preferably selected such that the viscosity of the mixture at a shear rate of 0 to 0.1 s -1between 0.1 Pa·s and 10 Pa·s and the viscosity of the mixture at a shear rate of 10 3 s -1 up to 10 4 s -1 < 0.01 Pa·s

[0053] The use of shear-thinning media, such as xanthan gum, PEO, HEC, or alginate, to inhibit sedimentation is generally known from the articles by Malcolm A. Faers and Grahman R. Kneebone, “Application of rheological measurements for probing the sedimentation of suspension concentrate formulations”, Pestic Sci 55:312-325 (1999); Cari A. Launiere et al., “Rheologically biomimetic cell suspensions for decreased cell settling in microfluidic devices”, Biomed Microdevices 13:549-557 (2011); or Bruno Arantes Moreira et al., “Analysis of suspension sedimentation in fluids with rheological shear-thinning properties and thixotropic effects”, Powder Technology 308 (2017) 290-297. Here, static application cases were examined in which the sedimentation of the objects during storage of the suspension had to be avoided while still maintaining a certain degree of flowability.

[0054] The shear-thinning medium is preferably selected from one or more substances of the group consisting of xanthan gum, alginates, polyethylene oxide (PEO), polyvinylpyrrolidone (PVP), carboxymethylcellulose, hydroxymethylcellulose, guar gum, locust bean gum.

[0055] The molecules that cause the shear-thinning properties are preferably dissolved in the carrier liquid. The mixture of the carrier liquid and the shear-thinning medium is therefore preferably a solution, less preferably an emulsion.

[0056] Suitable carrier fluids include a pure solvent, a solution, or an emulsion, preferably water-based. Particularly preferably, the carrier fluid contains or consists of a phosphate-buffered salt solution (PBS). To adjust the pH of the carrier fluid, it may be advantageous for it to contain further additives such as HCl or NaOH.

[0057] Xanthan gum is advantageously included in the mixture as a shear-thinning medium in a proportion of more than 0.075 wt% and less than 0.55 wt% based on the mixture. This applies particularly when using PBS as the carrier fluid or a carrier fluid containing PBS.

[0058] Xanthan gum exhibits particularly strong shear thinning even at concentrations of 0.01% to 0.5% within a viscosity range of 0.0015 to 80 Pa·s. This shear thinning property allows the dispersion to be used in the same fluidic system at a very wide flow rate range, from a few µL / min to several mL / min, without causing sedimentation or requiring excessively high pressures. Furthermore, xanthan gum is biocompatible and can be combined with buffers used in biology, such as PBS, without significantly altering its rheological properties relevant to such applications. Due to its rheological similarity, xanthan gum can even serve as a blood model in biological experiments.

[0059] The fluidic structure is preferably part of a microfluidic system by means of which the objects are subjected to one or more process steps consisting of detection, analysis, singulation and dispensing.

[0060] Detection encompasses optical detection using absorption, transmitted, and reflected light methods, as well as fluorescence-based optical methods. The invention utilizes the property of shear thinning to ensure that the objects to be detected are reliably and precisely transported within the same object plane. The optical measuring device is adjusted for maximum sensitivity within this object plane.

[0061] The analysis includes, in particular, counting and measuring the transport velocity. The invention utilizes the property of shear thinning in such a way that all objects are transported in one plane, and optical obscuration of one object by another is not possible. Furthermore, it is advantageous for measuring the transport velocity that the objects are all transported in one plane, namely precisely the one that lies at the maximum of the flow profile. This results in two further advantages. The actual flow rate in the channel can be clearly deduced from the object velocity. The dependence on object size is minimal in this case, in contrast to the situation with Newtonian fluids and transport at the equilibrium position on one flank of the flow profile. Measuring the flow velocity is therefore advantageous with regard to process control.

[0062] Singulation comprises dispensing or (briefly) switching the flow at a channel branch to transport target objects into a collection channel and all other objects and the remaining liquid, e.g., into a waste channel. Dispensing, in turn, comprises dispensing defined quantities of liquid from the cartridge, e.g., into wells of a microtiter plate. The invention utilizes the property of shear thinning in such a way that the objects no longer leave the position at the maximum flow velocity, thus enabling an ideal prediction of the object's position along the channel. This ensures that the actuation for singulating the target object can be triggered at the optimal time at a later position ("downstream") in the channel. A further advantage arises from the fact that sedimentation is prevented both after dispensing and in the collection channel.After dispensing, the fluid is at rest and its viscosity increases again. In the collecting channel, flow and shear rates are generally much lower. Nevertheless, sedimentation can be prevented due to the viscoelastic force towards the center of the channel. If the flow in the collecting channel stops completely, the viscosity increases further, and sedimentation is still prevented.

[0063] The mixing ratio of the carrier fluid and the shear-thinning medium, on the one hand, and the flow rate in the channel, on the other, are preferably set such that the objects are transported through the channel after passing through an inlet section at the latest in the center of the channel. The inlet section is defined as a segment of the channel that begins at the outlet and has a length in the direction of transport that corresponds to 5 to 10 times the minimum extent of the channel cross-section or the outlet, i.e., generally the extent in the depth direction d. It should be noted that transport towards the center is asymptotic. In this context, transporting the objects in the center of the channel means transporting the objects at a minimum distance of 10% of the minimum extent of the channel cross-section from all walls. This practically prevents sedimentation within the channel.

[0064] Furthermore, the mixing ratio of the carrier fluid and the shear-thinning medium, on the one hand, and the flow rate in the channel, on the other hand, are preferably adjusted such that the objects are transported through the channel at a uniform speed after an inlet section. Uniform here means, regardless of the object size, as long as its largest diameter does not exceed one-third of the smallest dimension of the channel, a maximum speed difference of 2% or less between the fastest and slowest objects.

[0065] Preferably, the fluidic structure is pre-filled with a buffer solution, at least along the circumference of the channel up to the outlet opening, before the mixture is introduced. This further reduces the risk of bubbles becoming trapped in the liquid when the mixture is introduced into the reservoir.

[0066] For example, the objects are biological cells.

[0067] In principle, additional additives can be introduced alongside the shear-thinning medium to improve cell conditions. An isotonic buffer solution is particularly preferred.

[0068] Isotonic here means that the tonicitic ratio, i.e. the osmotic pressure, between the buffer solution and the fluid in the cells is the same.

[0069] The method according to the invention is, for example, a method for performing flow cytometry or cell measurement, but is not limited to these. Other areas of application generally include: - All (micro)fluidic applications in which dispersions are used that could sediment or become separated during the process and - (micro-)fluidic applications where focusing of the objects and particularly gentle transport of the analytes in the center of the channel are necessary; and in particular: - Flow cytometric counting of biological objects such as CTCs, leukocytes, bacteria, Legionella, etc. in various carrier fluids such as blood, buffer systems, drinking water, bathing water, - Flow cytometric analysis of water samples with regard to various microparticles, such as microplastics and nanoparticles, - Sample focusing in imaging techniques (including flow cytometry and microscopy) without complex channel structures and - Analysis of sensitive analytes, for example biological objects such as cells or microorganisms, which take place in fluidic systems and should be non-destructive.

[0070] Further features and advantages of the invention are explained below with reference to an exemplary embodiment and the figures. These show: Fig. 1 a schematic representation of a microfluidic system in the area of ​​sample supply; Fig. 2 a schematic representation of another embodiment of the microfluidic system; Fig. 3 a count rate diagram; Fig. 4. A diagram to illustrate the relationship between viscosity and shear rate and Fig. 5 Diagram illustrating the dependence of shear thinning on additives in the dispersion.

[0071] In Fig. Figure 1 shows a partial and schematic embodiment of a microfluidic system with a base plate or substrate 10. The substrate 10 is plate-shaped and defines a coordinate system in three mutually perpendicular directions: a depth direction d, a height direction h, and a width direction w.

[0072] The substrate 10 incorporates a fluidic structure for the low-loss and gentle storage and transport of objects in a dispersion along a transport direction 12. The transport direction 12 preferably corresponds to the orientation shown in the diagram. Fig. 1 shown, when the microfluidic system is used as intended, it is in conjunction with the direction of gravity 13 and therefore runs parallel to the vertical direction h.

[0073] The fluidic structure comprises a reservoir 14 in the form of a depression and a channel 16 in the form of a trench or groove, wherein the thickness D of the substrate limits the maximum depth of the structures.

[0074] A lid or cover is not shown here. This is laminated onto the open top surface of the substrate 10 to close the reservoir 14 and the channel 16, except for the transport direction 12.

[0075] Reservoir 14 has an outlet 18 leading to channel 16, which is shown as a dashed plane at the transition between reservoir 14 and channel 16. The transport direction 12 is, by definition, perpendicular to the outlet 18. Reservoir 14 has a cross-sectional area perpendicular to the transport direction, which is shown here as an example dashed plane 20 at the vertical position where, viewed along the transport direction 12, reservoir 14 transitions from a section 22 with a constant cross-sectional area to a tapered section 24. Within the tapered section 24, the cross-sectional area tapers along the transport direction from a maximum value F max at the position of the upper level 20 to a minimum value F minat the position of the outlet opening 18. More precisely, the cross-sectional area of ​​the reservoir 14 decreases in the narrowing section 24 along the transport direction, viewed in the lateral direction w.

[0076] The cross-sectional area F K The cross-sectional area of ​​the channel 16 perpendicular to the transport direction 12 is equal to the cross-sectional area of ​​the mouth opening 18 and thus equal to F min .

[0077] The cross-sectional area of ​​reservoir 14 in the region of the maximum value F max has a minimum extent in the depth direction d and a maximum extent in the width direction w, wherein the aspect ratio between the maximum extent and the minimum extent of the cross-sectional area is in the range of 1 to 30, preferably in the range of 3 to 20.

[0078] The reservoir is bounded in the lateral direction w by a first wall 26, which defines a normal direction 28 at every point. In the tapered section 24, the normal direction 28 forms an angle α with the transport direction 12 or the vertical direction h, which lies in the range of 95°, preferably 100°, to 170°, preferably 165°. The normal direction 28 on the first wall 26 always has a component pointing in the lateral direction w and a component pointing in the vertical direction h opposite to the direction of gravity 13. That is, the first wall 26 has neither a horizontal nor a vertical orientation. The limiting values ​​of the angle α therefore ensure that, on the one hand, there is a sufficient inclination everywhere along this wall 26 to prevent sedimentation, and on the other hand, that the reservoir provides sufficient volume with a low overall height.

[0079] Unlike in this embodiment, the first wall in the tapered section could, for example, have a curved profile instead of a linear one, even within the angular limits of the surface normal.

[0080] The total volume of reservoir 14 consists of the volume of the tapered section 24 and the volume of section 22 with constant cross-sectional area F. max together. The first wall 26, which bounds the reservoir in the width direction w, runs parallel to the transport direction 12 in the section 22 with constant cross-sectional area. The first wall 26 has a kink at the transition between the tapered section 24 and the section 22 with constant cross-sectional area, which in practice, as in all other respects, is advantageously rounded to a greater or lesser degree depending on the dimension.

[0081] A second wall 30, which bounds reservoir 14 in the width direction w, lies opposite the first wall 26. This second wall 30 runs in the height direction h and thus parallel to the transport direction. It therefore does not contribute to the narrowing.

[0082] The reservoir 14 has a wall 32 opposite the outlet opening 18 with respect to the vertical direction h or transport direction 12, in which a filling opening 34 is arranged. The outlet opening 18 and the filling opening 34 are arranged transversely to the transport direction 12, i.e., offset from each other in the lateral direction w, and do not overlap in a projection in the transport direction 12 or vertical direction h. A liquid or dispersion, which is introduced into the reservoir 14, for example, by means of a pipette through the offset filling opening 34, therefore falls onto the first wall 26 in the tapered section 24 and flows down along this in a controlled manner to the outlet opening 18, while the air is gradually displaced from there.

[0083] The microfluidic structure of another embodiment according to Fig. 2 has a first reservoir 14 in a substrate 10, as described above with reference to Fig. The system is described in Figure 1 and includes a channel 16. The channel 16 comprises a connecting section 35, a first valve 36, a branch 37, a buffer channel 38, a second valve 39, a transition 40, and a functional channel (not shown in this illustration) which is fluidically connected to the transition 40 in a lower level on the substrate 10. The buffer channel 38 has a meandering profile in sections to provide a sufficiently large volume for a reservoir of the dispersion while requiring minimal space on the substrate 10. On its side opposite the outlet 18, the buffer channel 38 has an interface to which a pump can be connected directly or indirectly. The buffer channel 38 has a width of 1 mm and a channel depth of 1.5 mm. The storage volume of the meandering channel is 500 µl.

[0084] The functional channel is, for example, a cytometry measurement channel, with a channel width of 0.3 mm and a channel height of 0.05 mm.

[0085] The invention is described below using a method example with reference to Fig. 2 explained.

[0086] First, the fluidic structure is pre-filled with a buffer solution via interface 42. Valves 36 and 39 are initially opened while the buffer solution is pumped into buffer channel 38, into connecting section 35, and into the functional channel. The valves are then closed to ensure that both the functional channel and the connecting section are completely filled, thus guaranteeing bubble-free processing.

[0087] Subsequently, a mixture of a carrier fluid, a shear-thinning medium and the objects dispersed therein is introduced into the reservoir 14 through the filling opening 34 in the manner described above, without bubbles.

[0088] The first valve 36 is then opened, and the mixture is drawn or "drawn up" from reservoir 14 into buffer channel 38 by means of an external pump connected to interface 42. The pre-filled buffer solution in this channel branch is thereby lost. The typical flow rate for this process is approximately 360 µL / min, and the transport velocity is accordingly about 4 mm / s.

[0089] Subsequently, the first valve 36 is closed again and the second valve 39 is opened. When the pumping direction of the pump connected to interface 42 is then reversed, the mixture from the buffer channel 38 is forced through the transition 40 into the functional line below. Since all channels are filled without bubbles from the outset, the mixture to be processed is also conveyed into the functional channel without bubbles. The typical flow rate for this conveying and measuring process is, for example, approximately 5 µL / min, and the corresponding transport velocity is approximately 5.6 mm / s.

[0090] The invention is explained below using an exemplary mixture.

[0091] The inventors have successfully used xanthan gum in a device with a microfluidic structure for the automated immunomagnetic enrichment, detection, and isolation of circulating tumor cells. The immunomagnetically enriched cells were analyzed in a PBSE buffer with 0.125% xanthan gum according to a fluidic flow schedule in a microfluidic cartridge according to the invention, within a flow cytometer, and subsequently individually suspended. Flow velocities ranging from nearly 0 µm / s in the reservoir to >> 10 mm / sec in the functional channel, in this case a measurement channel with a cross-sectional area of ​​0.06 mm x 0.5 mm, were achieved. The cell recovery rate was significantly increased from below 50% to over 90% through the use of xanthan gum.

[0092] In Fig. Figure 3 shows a diagram of the cell count rates detected in the measuring channel. As can be seen from the diagram, the number of measured cells remains constant over time within the measurement error; that is, with the set mixture and the shear rates established in the fluid structure, no measurable cell loss due to sedimentation occurs at any point.

[0093] Fig. Figure 4 shows a diagram illustrating the relationship between viscosity and shear rate for various mixing ratios of a carrier fluid containing 9.55% PBS and the shear-thinning medium xanthan gum. As can be seen, xanthan gum exhibits significant shear thinning even at concentrations of 0.01% to 0.5% within a viscosity range of 0.0015 to 80 Pa·s. In particular, concentrations of >0.1% xanthan gum in this carrier fluid already show a sufficient shear rate contrast, i.e., a viscosity difference of at least two orders of magnitude within a shear rate interval of 0.1 s⁻¹ to 1000 s⁻¹.

[0094] The dependence of the shear-thinning property of xanthan gum on the addition of a PBS buffer is shown from Fig.Figure 5 shows that if the carrier fluid contains no additives and consists only of water, a significant effect is already achieved at a xanthan gum concentration of 0.05%. To achieve a comparable shear rate contrast, the xanthan gum concentration must be adjusted when, for example, adding 9.55% PBS to the carrier fluid. Reference symbol list 10 substrate 12. Transport direction 13 Direction of gravity 14 Reservoir 16-channel Level 18, estuary 20 Level, cross-sectional area 22 Section with constant cross-sectional area 24 Rejuvenation section 26 first wall 28 Normal, normal direction 30 second wall 32 opposite wall 34 Filling opening 35 Connecting section 36 first valve 37 Junction 38 Buffer channel 39 second valve 40 Transition 42 Interface d Depth direction hw Altitude direction Latitude direction α angle QUOTES INCLUDED IN THE DESCRIPTION

[0000] This list of documents cited by the applicant was automatically generated and is included solely for the reader's convenience. The list is not part of the German patent or utility model application. The DPMA accepts no liability for any errors or omissions. Cited non-patent literature

[0000] Papers by Malcom A. Faers and Grahman R. Kneebone “Application of rheological measurements for probing the sedimentation of suspension concentrate formulations,” Pestic Sci 55:312-325 (1999); Cari A. Launiere et al “Rheologically biomimetic cell suspensions for decreased cell settling in microfluidic devices”, Biomed Microdevices 13:549-557 (2011

[0053] Bruno Arantes Moreira et al „Analysis of suspension sedimentation in fluids with rheological shear-thinning properties and thixotropic effects“, Powder Technology 308 (2017) 290-297

[0053]

Claims

[1] Fluidic structure for low-loss and gentle storage and transport of objects in a dispersion, wherein the fluidic structure has a reservoir (14) and a channel (16) for the dispersion, wherein the reservoir (14) has a filling opening (34) and an outlet opening (18) to the channel (16), wherein a transport direction (12) is defined perpendicular to the outlet opening (18), wherein the reservoir (14) has a cross-sectional area (20) perpendicular to the transport direction (12), wherein the reservoir (14) has at least one tapered section (24) in which the cross-sectional area (20) decreases along the transport direction (12) from a maximum value F max to a minimum value F min < F max funnel-shaped tapering, with the minimum value F min corresponds to the cross-sectional area of ​​the mouth opening (18). [2] Fluidic structure according to claim 1, characterized by , that the ratio of the maximum value to the minimum value is 5 < Fmax / F min < 10 6 . [3] Fluidic structure according to claim 1 or 2, characterized by , that the reservoir (14) has a volume V R and the channel (16) has a volume V K exhibit, where the ratio of the reservoir volume to the channel volume is: V K < V R and where the volume of the reservoir V R in the range of 0.1 ml to 10.0 ml. [4] Fluidic structure according to one of the preceding claims, characterized by , that the fluidic structure is formed in a plate-shaped substrate (10) which defines a coordinate system with a depth direction (d), a height direction (h) and a width direction (w) in three mutually perpendicular directions, wherein the reservoir (14) is formed by a depression and the channel (16) by a channel in the substrate (10) and wherein the transport direction (12) coincides with the height direction (h). [5] Fluidic structure according to claim 4, characterized by , that the cross-sectional area (20) of the reservoir (14) is in the range of the maximum value F max has a minimum extent in the depth direction (d) and a maximum extent in the width direction (w), wherein the aspect ratio between the maximum extent and the minimum extent of the cross-sectional area (20) is in the range of 1 to 30, preferably in the range of 3 to 20. [6] Fluidic structure according to one of claims 4 to 5, characterized by , that the cross-sectional area (20) of the reservoir (14) decreases in the narrowing section (24) along the transport direction (12) in the lateral direction (w). [7] Fluidic structure according to claim 6, characterized by, that at least one first wall (26) bounding the reservoir (14) in the width direction (w) defines a normal direction (28) at every point of the first wall (26) which in the tapered section (24) encloses an angle α with the transport direction (12) for which 95° < α, preferably 100° < α, and 170° > α, preferably 165° > α. [8] Fluidic structure according to one of the preceding claims, characterized by , that the reservoir has a wall (32) opposite the outlet opening (18) with respect to the transport direction (12), in which the filling opening (34) is arranged, wherein the outlet opening (18) and the filling opening (34) are arranged transversely to the transport direction. [9] Fluidic structure according to claim 8, characterized by , that the outlet opening (18) and the filling opening (34) do not overlap in a projection in the transport direction (12). [10] Fluidic structure according to one of the preceding claims, characterized by, that the channel (16) has a buffer channel (38) or a function channel or a buffer channel (38) and a function channel. [11] Method for low-loss and gentle storage and transport of objects in a dispersion, comprising the steps: - Providing a fluidic structure according to any one of claims 1 to 10, - Introducing a mixture of a carrier fluid, a shear-thinning medium and the objects dispersed therein into the reservoir (14), - Transporting at least part of the mixture from the reservoir (14) through the channel (16) at a flow rate, - wherein the mixing ratio of the carrier fluid and the shear-thinning medium on the one hand and the flow rate in the fluidic structure on the other hand are set such that the mixture has a sufficiently high viscosity at rest or at lower transport speeds to prevent the objects from sedimenting, and that at higher transport speeds the objects in the mixture are moved from the channel wall to the center of the channel and transported further there due to the shear-thinning behavior of the mixture. [12] Method according to claim 11, characterized by , that the viscosity difference in a shear rate interval of 0.1 s -1 up to 1000 s -1 at least two orders of magnitude. [13] Method according to claim 11 or 12, characterized by , that the concentration of the shear-thinning medium in the mixture is chosen such that the viscosity of the mixture at a shear rate of 0.01 s -1between 0.1 Pa·s and 10 Pa·s, and the viscosity of the mixture at a shear rate of > 10,000 s -1 less than 0.01 Pa·s. [14] Method according to any one of claims 11 to 13, characterized by , that the shear-thinning medium is selected from one or more substances of the group consisting of xanthan gum, alginates, polyethylene oxide (PEO), polyvinylpyrrolidone (PVP), carboxymethylcellulose, hydroxymethylcellulose, guar gum, locust bean gum. [15] Method according to any one of claims 11 to 14, characterized by that the carrier fluid contains or consists of a phosphate-buffered saline solution (PBS). [16] Method according to claim 15, characterized by , that xanthan gum is present in the mixture as a shear-thinning medium in a proportion of more than 0.075 wt% and less than 0.55 wt% based on the mixture. [17] Method according to any one of claims 11 to 16, characterized bythat the fluidic structure is part of a microfluidic system by means of which the objects are subjected to one or more process steps of detection, analysis, singulation and dispensing. [18] Method according to any one of claims 11 to 17, characterized by , that the mixing ratio of the carrier fluid and the shear-thinning medium on the one hand and the flow rate in the channel on the other hand are set so that the objects are transported through the channel at the latest after an inlet section in the center of the channel. [19] Method according to any one of claims 11 to 18, characterized by, that the mixing ratio of the carrier fluid and the shear-thinning medium on the one hand and the flow rate in the channel on the other hand are set such that the objects are transported through the channel at a uniform transport speed, whereby the objects, regardless of their size, as long as their largest diameter is not more than one third of the smallest dimension of the channel, have a maximum velocity difference of 2% or less.

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