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 comprising a reservoir (14) and a channel (16), and to a method for the low-loss and gentle storage and transport of objects in a dispersion. The method comprises the steps of: providing a fluidic structure, introducing a mixture consisting of a carrier liquid, a shear-thinning medium and the objects dispersed therein into the reservoir (14), transporting at least some of the mixture from the reservoir (14) through the channel (16) at a flow rate, wherein the mixing ratio of the carrier liquid and the shear-thinning medium, and the flow rate in the fluidic structure are set such that, at rest or at a lower transport speed, the mixture has a sufficiently high viscosity which prevents the objects from sedimenting, and that, at a higher transport speed, the objects in the mixture are moved from the channel wall to the middle of the channel due to the shear thinning behaviour of the mixture and are transported further there.

Description

[0001] Fluidic structure and method for low-loss and gentle storage and transport of objects in a dispersion

[0002] Description

[0003] 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 pl to 10 ml.

[0004] 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.

[0005] 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 laminated onto the substrate to seal these grooves and depressions. The base plates are made of plastic using injection molding or embossing processes.

[0006] I 1184 WO molded and the cover foils were fluid-tightly connected to the base plates by adhesive or welding processes.

[0007] 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 pm 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.

[0008] 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.

[0009] 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

[0010] In I 1184 WO, on the one hand, the sedimentation distances are shorter, and on the other hand, due to the large surface area to volume ratio, a proportionally larger surface area is available for adhesion. Furthermore, the sedimentation and adhesion of objects, particularly in microfluidic systems, can lead to blockages of the microchannels.

[0011] 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.

[0012] 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.

[0013] 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

[0014] I 1184 WO to provide 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.

[0015] The problem is solved by a fluidic structure having the features of claim 1 and a method having the features of claim 11.

[0016] 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. Furthermore, the reservoir has at least one tapered section in which the cross-sectional area tapers in a funnel shape along the transport direction from a maximum value Fmax to a minimum value Fmin < Fmax, where the minimum value Fmin corresponds to the cross-sectional area of ​​the outlet opening.

[0017] The channel geometry according to the invention ensures easy filling of the reservoir due to the maximum cross-section at the inlet. The cross-sectional area of ​​the channel FK perpendicular to the transport direction is preferably equal to the cross-sectional area of ​​the outlet opening, i.e., preferably equal to Fmin, and in any case smaller than the maximum cross-sectional area Fmax. Due to the funnel-shaped narrowing from the maximum cross-section to the cross-section of the channel, a continuous transition is provided, during which the flow velocity in the transport direction constantly increases. This alters the shear forces and wall effects acting in the liquid in a controlled manner, which, in conjunction with a shear-densing behavior of the liquid adapted to this, reduces sedimentation even under static conditions.

[0018] I 1184 WO effectively prevents dispersion in the fluidic structure as well as in the dynamic transport case.

[0019] Preferably, the ratio of the maximum value to the minimum value is 5 < Fmax / Fmin < 10^.

[0020] Fmax largely determines the reservoir volume, while Fmin determines the maximum flow velocity in the transition zone between the reservoir and the channel. The lower limit 5 < Fmax / Fmin of the ratio is determined, firstly, by a practical minimum reservoir size, as otherwise filling the reservoir with liquid would be very difficult, and secondly, by the fact that Fmin cannot be chosen to be arbitrarily large due to the generally small volumes involved.

[0021] The upper limit F max / F min < 10 6Conversely, the cross-sectional area of ​​the channel FK is determined by its size, which is limited to minimum dimensions of 10 µm x 10 µm. Such small channels are just about suitable for handling bacteria. At the same time, larger reservoirs become impractical, as processing larger volumes at realistic pressures would take far too long.

[0022] The reservoir preferably has a volume VR and the channel a volume VK, wherein the ratio of the volume of the reservoir to the volume of the channel VK < VR, and wherein the volume of the reservoir VR is 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.

[0023] The fluidic structure is preferably formed in a plate-shaped substrate which has a coordinate system in three mutually perpendicular directions with a depth direction d, a height direction h and a width direction w.

[0024] I 1184 WO defined, 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 vertical direction.

[0025] 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.

[0026] The cross-sectional area of ​​the reservoir in the region of the maximum value Fmax therefore also has its minimum extent in the depth direction and a maximum extent in the width direction, 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.

[0027] Preferably, the cross-sectional area of ​​the reservoir decreases in the tapered section along the transport direction in the lateral direction.

[0028] The change in cross-section can be manufactured most accurately in this direction.

[0029] 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, hold true.

[0030] The transport direction of the microfluidic system preferably coincides with the direction of gravity when used as intended. 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, i.e.,

[0031] In I 1184 WO the first wall has no horizontal orientation at any point. The upper limit of the angle o of 170° and preferably of 165°, causes a sufficient inclination along this wall to reduce the risk of sedimentation here.

[0032] 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.

[0033] The tapered section itself can provide a significant portion of the reservoir's volume. Upstream of the tapered 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 Fmax, to increase the volume to the required size without having to accept an excessively wide reservoir. During a tapered section, the angle α is < 90°, which is not entirely impossible, as sedimentation cannot occur on such surfaces. For better surface utilization on the substrate, the first wall, which bounds the reservoir laterally, preferably runs parallel to the direction of flow 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.

[0034] A second wall, which defines the reservoir's width, lies opposite the first wall. This second wall can optionally run parallel to the direction of transport and thus not contribute to the narrowing, or it can also have a normal direction that forms an angle α > 90° with the direction of transport in the narrowing section, thus also contributing to the narrowing.

[0035] I 1184 WO Advantageously, the reservoir has a filling opening on a wall opposite the direction of transport of the outlet opening, wherein the outlet opening and the filling opening are arranged transversely to the direction of transport.

[0036] The outlet opening and the filling opening preferably do not overlap in a projection in the direction of transport.

[0037] 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.

[0038] 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 and to push a fluid (buffer solution, dispersion) into or out of the buffer channel.

[0039] Preferably, the buffer channel has a cross-sectional area Fp, where the ratio of the maximum value Fmax to the cross-sectional area Fp is: 5 < Fmax / FP < 1000. In a preferred embodiment of the invention, the buffer channel has a meandering shape, at least in sections, to create a

[0040] I 1184 WO to create a sufficiently large volume for a supply of the dispersion and at the same time be able to be arranged on the substrate in a space-saving manner.

[0041] In another preferred embodiment, the channel comprises a functional channel with a cross-sectional area FM, wherein the ratio of the maximum value Fmax to the cross-sectional area FM is preferably: 100 < Fmax / FM < 10 6 .

[0042] A "functional channel" is a channel section following the main channel in which the objects are subjected to process steps such as detecting, analyzing, singulating or dispensing, for example a measuring channel, detection channel or analysis channel or dgL.

[0043] Preferably, the maximum value Fmax of the cross-sectional area of ​​the reservoir is 10 mm². 2 up to 50 mm 2 Preferably, the cross-sectional area of ​​the buffer channel Fp is 0.1 mm². 2 up to 5 mm 2Preferably, the cross-sectional area of ​​the functional channel FM is 100 pm. 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.

[0044] 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.

[0045] The fluidic structure advantageously includes one or more valves that selectively open or close sections of the channel. The valves allow control of the movement of the dispersion or other fluids within the fluidic structure.

[0046] I 1184 WO can 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.

[0047] 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:

[0048] Providing a fluidic structure as described above,

[0049] Introducing a mixture of a carrier fluid, a shear-thinning medium, and the objects dispersed therein into the reservoir; transporting at least a 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-thickening behavior of the mixture.

[0050] 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.

[0051] I 1184 WO Hereinafter, without limitation to specific applications, the term dispersion refers to 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. Dispersion thus includes both suspensions and emulsions. For the purposes of this document, the carrier liquid may be (except for tolerable impurities) a pure liquid, a liquid solution, or itself already a dispersion.

[0052] 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).

[0053] Examples of the investigational dispersion are:

[0054] 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;

[0055] Milk: Droplet emulsion with slightly shear-thinning properties. However, other target objects, such as bacteria, may be dissolved in the milk;

[0056] A homogeneous shear-viscous medium, such as a xanthan gum solution, which is used as a transport medium for the cells;

[0057] Dispersions are a type of dispersion in which cells are contained within droplets, which are themselves suspended in a shear-thinning medium. This is used, for example, to create different environments within the droplet for individual cells and then to sort them based on their response to these environments.

[0058] I 1184 WO The channel geometry and the set conveying or flow rate determine the transport velocities and thus the shear forces in the fluidic structure. In the reservoir, the dispersion is at rest or, during transport, moves at a transport velocity typically ranging from 0 pm / s to 10 mm / s, relative to areas of the fluidic structure with a cross-sectional area Fmax. In the channel, if it is designed as a buffer channel, the transport velocity is typically between 10 pm / s and 25 mm / s. Higher transport velocities in the range of mm / s are used during filling, and lower transport velocities in the range of 10,000–15,000 pm / s are used during transport into the functional channel. In the functional channel, the velocity ranges from 1 mm / s to 1,000 mm / s.

[0059] The properties of shear-thinning media are already strongly pronounced at very low concentrations.

[0060] 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.

[0061] In Newtonian fluids, there are hydrodynamic forces that, on the one hand, displace objects 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 opposite force that pushes objects from the center of the channel, where the flow velocity is highest and the curvature is greatest.

[0062] I 1184 WO Displace the object in the direction of the lesser curvature of the river profile. At a certain distance from the canal wall and the center of the canal (locations of highest flow velocity), these two forces are in equilibrium and objects are transported at this distance from the wall ("equilibrium position").

[0063] 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.

[0064] 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.

[0065] 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 currents according to the principle of...

[0066] I 1184 WO hydrodynamic focusing is achieved. This effort is no longer necessary, since in the case of the inventive method, the objects are transported solely by the non-Newtonian fluid in the center, even without limiting elements. If enveloping flows are required for processing in other ways, for example, to add further substances, this is still possible without affecting the described effect of focusing and sedimentation prevention.

[0067] 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.

[0068] 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 if 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 if, at higher transport speeds within small channel cross-sections, the objects are transported within the mixture due to the properties described above.

[0069] In 1184 WO, shear-thinning media are moved from the channel wall towards the center of the channel and transported further there. This prevents sedimentation even in the moving liquid. The relative values ​​"lower transport velocity" and "higher transport velocity" refer only to each other; that is, the statement is simply that higher transport velocity > lower transport velocity. The absolute values ​​of both lie within the ranges specified above.

[0070] 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.

[0071] In particular, the concentration of the shear-thinning medium in the mixture is preferably chosen 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.

[0072] In principle, the use of shear-thinning media, such as xanthan gum, PEO, HEG, or alginate, to inhibit sedimentation is supported by, for example, the articles by Malcolm A. Faers and Grahman R. Kneebone, “Application of rheological measurements for probing the sedimentation of suspension concentrate formulations,” Pestic Science 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.

[0073] I 1184 WO “Analysis of suspension sedimentation in fluids with rheological shear-thinning properties and thixotropic effects”, Powder Technology 308 (2017) 290-297. Here, static applications were investigated in which the sedimentation of objects during suspension storage had to be avoided while still maintaining a certain degree of flowability.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] 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.

[0078] Xanthan gum otherwise exhibits particularly strong shear thinning even at concentrations of 0.01% to 0.5% in a viscosity range of 0.0015 to

[0079] I 1184 WO 80 Pa s. The shear thinning property allows the dispersion to be used in the same fluidic system over a very wide flow rate range, from a few pL / 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.

[0080] 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.

[0081] 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 in such a way that the objects to be detected are reliably and precisely transported in the same object plane. The optical measuring device is adjusted for maximum sensitivity in this object plane.

[0082] 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

[0083] The size of the object is minimal in this case, unlike 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 for process control.

[0084] 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 directed towards the center of the channel. If the flow in the collecting channel stops completely, the viscosity increases further, and sedimentation is still prevented.

[0085] Particularly preferred are 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, such that the objects are transported through the channel after passing through an inlet section in the center of the channel at the latest. The inlet section is defined as a segment of the channel that begins at the outlet and has a length in the transport direction that is 5 to 10 times the

[0086] I 1184 WO corresponds to the minimum extent of the channel cross-section or the mouth opening, which is generally the extent in the depth direction d. It must be taken into account that transport towards the center occurs asymptotically. In this sense, transporting 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. In practice, sedimentation within the channel is then impossible.

[0087] 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.

[0088] 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.

[0089] For example, the objects are biological cells.

[0090] In principle, additional additives can be introduced alongside the shear-thinning medium to improve cell conditions. An isotonic buffer solution is particularly preferred.

[0091] 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.

[0092] I 1184 WO The method according to the invention is, for example, a method for performing flow cytometry or cell measurement, but is not limited to this. Other areas of application are generally:

[0093] - All (micro)fluidic applications in which dispersions are used that could sediment or become separated during the process and

[0094] - (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:

[0095] - 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,

[0096] - Flow cytometric analysis of water samples with regard to various microparticles, such as microplastics and nanoparticles,

[0097] - Sample focusing in imaging techniques (including flow cytometry and microscopy) without complex channel structures and

[0098] - Analysis of sensitive analytes, for example biological objects such as cells or microorganisms, which take place in fluidic systems and should be non-destructive.

[0099] Further features and advantages of the invention are explained below with reference to an exemplary embodiment and the figures. These show:

[0100] I 1184 WO Figure 1 a schematic representation of a microfluidic system in the area of ​​sample supply;

[0101] Figure 2 shows a schematic representation of another embodiment of the microfluidic system;

[0102] Figure 3 shows a count rate diagram;

[0103] Figure 4 is a diagram illustrating the relationship between viscosity and shear rate.

[0104] Figure 5 Diagram illustrating the dependence of shear thinning on additives in the dispersion.

[0105] 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.

[0106] 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 coincides with the direction of gravity 13, as shown in the orientation according to Figure 1, when the microfluidic system is used as intended, and therefore runs parallel to the vertical direction h.

[0107] 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.

[0108] I 1184 WO A lid film or cover is not shown here. This is laminated onto the open top of the substrate 10 to close the reservoir 14 and the channel 16 except for the transport direction 12.

[0109] 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 decreases along the transport direction from a maximum value Fmax at the position of the upper plane 20 to a minimum value Fmin at the position of the outlet 18. More precisely, the cross-sectional area of ​​reservoir 14 decreases in the tapered section 24 along the transport direction in the lateral direction w.

[0110] The cross-sectional area FK of the channel 16 perpendicular to the transport direction 12 is equal to the cross-sectional area of ​​the outlet opening 18 and therefore equal to Fmin.

[0111] The cross-sectional area of ​​the reservoir 14 in the region of the maximum value Fmax 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.

[0112] The reservoir is bounded in the lateral direction w by the first wall 26, which defines a normal direction 28 at each point. In the tapered section 24,

[0113] In accordance with I 1184 WO, 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 limit 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.

[0114] 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.

[0115] The total volume of reservoir 14 comprises the volume of the tapered section 24 and the volume of section 22 with constant cross-sectional area Fmax. The first wall 26, which bounds the reservoir in the width direction w, runs parallel to the transport direction 12 in section 22 with constant cross-sectional area. At the transition between the tapered section 24 and section 22 with constant cross-sectional area, the first wall 26 has a kink, which, in practice, as with all other depicted internal and external angles of the fluidic structure, is advantageously rounded to a greater or lesser degree depending on the dimensions.

[0116] 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.

[0117] In Figure 1184, 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.

[0118] The microfluidic structure of a further embodiment according to Figure 2 comprises a first reservoir 14 in a substrate 10, as described above with reference to Figure 1, and a channel 16. The channel 16 includes 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 plane 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 storage meander is 500 pl.

[0119] 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.

[0120] I 1184 WO The invention is explained below using a method example with reference to Figure 2.

[0121] 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, connecting section 35, and 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.

[0122] 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.

[0123] 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 pL / min, and the transport velocity is accordingly about 4 mm / s.

[0124] 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 pL / min, and the corresponding transport velocity is approximately 5.6 mm / s.

[0125] I 1184 WO The invention is explained below using an exemplary mixture.

[0126] 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 pm / 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.

[0127] 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.

[0128] 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

[0129] I 1184 WO Viscosity difference of at least two orders of magnitude in a shear rate interval from 0.1 s-1 to 1000 s-1.

[0130] The dependence of xanthan gum's shear-thinning property on the addition of a PBS buffer is shown in Figure 5. If the carrier fluid contains no additives and consists only of water, a significant effect is already observed 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.

[0131] I 1184 WO Reference List

[0132] 10 substrate

[0133] 12. Transport direction

[0134] 13 Direction of gravity

[0135] 14 Reservoir

[0136] 16-channel

[0137] Level 18, estuary

[0138] 20 Level, cross-sectional area

[0139] 22 Section with constant cross-sectional area

[0140] 24 Rejuvenation section

[0141] 26 first wall

[0142] 28 Normal, normal direction

[0143] 30 second wall

[0144] 32 opposite wall

[0145] 34 Filling opening

[0146] 35 Connecting section

[0147] 36 first valve

[0148] 37 Junction

[0149] 38 Buffer channel

[0150] 39 second valve

[0151] 40 Transition

[0152] 42 Interface d Depth direction h Height direction w Latitude direction

[0153] Q angle

[0154] I 1184 WO

Claims

Patent 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) tapers funnel-shaped along the transport direction (12) from a maximum value Fmax to a minimum value Fmin < Fmax, wherein the minimum value Fmin corresponds to the cross-sectional area of ​​the outlet opening (18).

2. Fluidic structure according to claim 1, characterized in that the ratio of the maximum value to the minimum value is 5 < Fmax / Fmin < 10^.

3. Fluidic structure according to claim 1 or 2, characterized in that the reservoir (14) has a volume VR and the channel (16) has a volume VK, wherein the ratio of the volume of the reservoir to the volume of the channel is: VK < VR and wherein the volume of the reservoir VR is in the range of 0.1 ml to 10.0 ml.

4. Fluidic structure according to one of the preceding claims, characterized in that the fluidic structure is formed in a plate-shaped substrate (10) which has a coordinate system in three mutually perpendicular directions with a depth direction (d), a height direction (h) and a I 1184 WO Latitude direction (w) is defined, 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 in that the cross-sectional area (20) of the reservoir (14) in the region of the maximum value Fmax 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 in that the cross-sectional area (20) of the reservoir (14) decreases in the tapered section (24) along the transport direction (12) in the lateral direction (w).

7. Fluidic structure according to claim 6, characterized in that at least one first wall (26) limiting the reservoir (14) in the width direction (w) defines a normal direction (28) at each 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 in that the reservoir has a wall (32) opposite the outlet opening (18) with respect to the transport direction (12), in which the I 1184 WO The filling opening (34) is arranged, wherein the outlet opening (18) and the filling opening (34) are arranged transversely to the direction of transport.

9. Fluidic structure according to claim 8, characterized in 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 in that the channel (16) has a buffer channel (38) or a functional channel or a buffer channel (38) and a functional channel.

11. Method for low-loss and gentle storage and transport of objects in a dispersion, comprising the following steps: Providing a fluidic structure according to one of claims 1 to 10, introducing a mixture of a carrier fluid, a fluid-diluting 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 at rest or at lower transport speeds has a sufficiently high viscosity 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. I 1184 WO 12. Method according to claim 11, characterized in that the viscosity difference is 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 in that the concentration of the shear-thinning medium in the mixture is selected such that the viscosity of the mixture at a shear rate of 0.01 s' 1 between 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 one of claims 11 to 13, characterized in 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 in that the carrier liquid contains or consists of a phosphate-buffered salt solution (PBS).

16. Method according to claim 15, characterized in that xanthan gum is 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. I 1184 WO 17. Method according to one of claims 11 to 16, characterized in that the fluidic structure is part of a microfluidic system by means of which the objects are subjected to one or more process steps consisting of detecting, analyzing, singulating and dispensing.

18. Method according to one of claims 11 to 17, characterized in 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 adjusted such 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 one of claims 11 to 18, characterized in 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 adjusted such that the objects are transported through the channel at a uniform transport speed, wherein the objects, regardless of their size, as long as their largest diameter is no more than one third of the smallest dimension of the channel, have a maximum speed difference of 2% or less. I 1184 WO

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