Cavity substrate with recesses for reversible uptake and microfluidic analysis, in particular counting, of biological cells, especially circulating tumor cells.

Abstract

The invention relates to a substrate (100) for the uptake and microfluidic analysis, in particular counting, of biological cells (40), especially circulating tumor cells (40), wherein the substrate (100) has cavities (120) on a top surface (110) for receiving the cells (40), wherein the walls (121, 122) and / or bottoms (123) of at least some cavities (120) have recesses (130), in particular pores, to facilitate the removal of uptaken cells (40) from the cavities (120). The invention further relates to a microfluidic device (200), and a method (500) for the uptake and microfluidic analysis of biological cells (40) with such a substrate (100), as well as a manufacturing method (600) of such a substrate (100).

Description

Prior ArtCirculating tumor cells (CTCs) are referred to as cells which separate from the primary tumor, penetrate into the peripheral blood stream and can pass over it into surrounding tissue. These cells, even despite surgery and therapy, present in the blood are referred to as "minimum residual disease (MRD)" and are considered to be the starting point for metastases and recurrences, as described, for example, in Meng et al. Circulating tumor cells in patients with breast cancer dormancy. Clin Cancer Res. 10, 2004, 24, pp. 8152-8162. Therefore, their detection and quantification (counting of the CTCs, CTC count) is relevant both for prognostic statements and for clinical management (Crispoganilli et al., Circulating tumor cells, disease progression, and survival in metastatic breast cancer. Semin. Oncol. 33, 2006, 3, pp. 9-14), in particular for the recognition of metastasis in an early stage and for efficiency control and adaptation during a therapy method.The quantification of CTCs from a so-called liquid biopsy, i.e. a blood sample of a tumor patient, has the advantage that it can be carried out as often as desired, since it is a minimally invasive intervention in the form of a blood collection. The greatest challenge here is the very small number of CTCs in relation to the blood cells-above all in the stage of MRD or in very early stages of metastasis. CTC detection therefore requires highly sensitive detection methods to detect the very small number of CTCs from millions of red and white blood cells early and efficiently.A possibility for quantifying CTCs from a blood sample by means of optical detection as free of loss as possible after fluorescent staining with tumor cell-specific antibodies of one type (e.g.: EpCAM-FITC) or of several types (e.g., EpCAM, cytokeratin, HER2-neu... ) is provided by a sedimentation array. This makes it possible to allow nucleated cells of a blood sample to sediment in cavities of a plane after erythrocyte lysis, in order to be able to detect and count these there in a spatially resolved manner by means of optical systems. In addition, the capture of the cells in these cavities allows the rinsing with different media and reagents in order to be able to rinse away excess lysis buffer, erythrocyte lysate, free dyes or the like and / or to rinse in further dye markings without the target cells changing their position or being rinsed out again. The laid-open specification DE 10 2021 203 897 A1 describes a microfluidic cartridge suitable for this purpose with a carrier substrate with a multiplicity of etched-in depressions as a sedimentation array, into which CTCs can be sedimented and subsequently analyzed and counted by means of their various fluorescent markings.Disclosure of the InventionAdvantages of the InventionAgainst this background, the invention relates to a substrate for receiving and microfluidic analysis, in particular counting of biological cells, wherein the substrate has cavities on an upper side for receiving the cells, characterized in that walls and / or bases of at least some cavities have recesses, in particular pores, for supporting removal of received cells from the cavities.To accommodate the cells, a sample containing the cells, in particular blood, can preferably be applied to the upper side of the substrate, preferably after a pretreatment of the blood, for example a lysis of blood constituents, in particular erythrocytes, and / or filtering of specific blood particles. In this case, the cells can sediment from the sample into at least some of the cavities. The blood may be whole blood. The sample can have been obtained by a, in particular immediately preceding, blood collection in a human or animal. The cells are in particular nucleated and / or nucleic acid-containing cells, preferably circulating tumor cells (CTCs), in particular circulating epithelial tumor cells. The substrate, which as described at the beginning can be used and referred to as a sedimentation array and can be based on the sedimentation array described in the laid-open specification DE 10 2021 203 897 A1 (referred to there as carrier substrate), is preferably silicon or a material comprising silicon. Alternatively, the substrate can comprise germanium, aluminum, titanium, tungsten or consist of one of these materials, for example. Cavities are to be understood as meaning, in particular, sack-shaped depressions on the upper side of the substrate. The substrate may be disposed in a microfluidic device for analysis of such cells. The invention thus also relates to a microfluidic device, in particular a microfluidic cartridge, comprising a substrate according to the invention. The invention furthermore relates to a method for producing such a substrate, wherein, in addition to producing the cavities, which can preferably be carried out, in particular in the case of silicon, by an anisotropic etching method, the recesses are formed in the cavities, in particular by porosification of at least a part of the substrate comprising the cavities concerned. A poration of the substrate is understood to mean, in particular, an etching process, in particular an electrochemical etching process into hydrofluoric acid-containing electrolytes or, alternatively, an electroless etching process into hydrofluoric acid-containing electrolytes in conjunction with oxidizing agents (for example nitric acid combined with acetic acid, for example as HNA=hydrofluoroacid (H), nitronic acid (N) and acetic acid (A)), whereby a substrate region, in particular a silicon region, is converted into a porous spongy material in a controlled manner. The thickness of the surface layer porousised in this way is largely freely selectable and well-defined in a time-controlled manner over the process time. As an alternative to poration of silicon, the substrate to be porated may, as described above, comprise, for example, germanium, aluminum, titanium, tungsten or consist of one of these materials.Due to the structures connected thereto, in particular nanostructures, the recesses in the walls and / or bases of the cavities advantageously ensure adhesion, in particular adhesion, to the surface, which has the advantage that the cells received can be easily removed from the cavities if necessary. It is furthermore advantageous that the cells taken up can be analyzed from all sides without adhesion and / or can interact comprehensively with other entities, in particular reagents, which facilitates processing of the cells in the cavities. The invention therefore also relates to a method for the analysis, in particular counting, of biological cells, in particular circulating tumor cells, with such a substrate, wherein at least some of the cells preferably taken up into the cavities via sedimentation are removed again from the cavities after a preferred analysis, in particular counting.In the simplest embodiment of the process according to the invention, the preferred sedimentation is again effected by gravity, as before. The force of gravity, which during the preferred sedimentation phase has let the cells sink into the cavities, is used by tilting, turning or inverting the substrate or entire microfluidic device or cartridge together with substrate to transport the cells out of the cavities following the changed direction of the force of gravity. In particular, the cells can be transferred into an aqueous phase initially above the surface of the substrate, where they can be picked up by a pipette or a syringe after a certain waiting time or transported by a flow to an outlet (and a pick-up station located there). The removal can take place in particular after completion of a counting of the recorded cells, for example in order to feed the cells outside the cavities to a continuing analysis depending on the counting requirement.The recesses, in particular pores, in the cavities have in particular a size which is smaller than the size of the cells to be taken up and analyzed, preferably smaller than typical circulating tumor cells, in particular smaller than epithelial cells. For example, the recesses, in particular the pores, have a size between 1 nm and 100 nm (corresponding to a nanopority) or a size of 0.1-1 μm (corresponding to a mesopority) or a size of 1-5 μm (corresponding to a macropority). The size of the recesses or pores can be determined in particular by the etching process (current density, electrolyte concentration, concentration of the oxidizing agent, doping level in the silicon).Preferably, at least some of the cavities each have more than one recess, in particular more than 10, preferably more than 100, very preferably more than 500 recesses. The recesses can be pores in particular. At least a part, in particular a layer, of the substrate comprising at least some of the cavities can be porousised for this purpose, in particular meso- and / or nano-porousised. This part of the substrate thus preferably has a sponge-like structure, through which channels created by the poration can pass, the ends of which opening into the cavities preferably form recesses according to the invention. In the case of porous silicon and materials having similar effects, the high biocompatibility is advantageously utilized in this case, wherein the porosity greatly inhibits an increase or adhesion of biological substances, in particular cells or proteins, and thus prevents "logging" or "biofouling". The cells accommodated in the cavities, which come into contact with the porous (silicon) base on which they come to lie and possibly also with the porous (silicon) substrates, do not build up any bonds to the contact surfaces, i.e. do not stick on, but rather remain movable in the depressions. They can thus also be conveyed out of the cavities again if necessary by the action of only small forces.According to a particularly advantageous development of the invention, at least one recess, preferably in each case at least one recess in a plurality of cavities, extends as a through channel as far as a second side of the substrate, in particular as far as an underside of the substrate opposite the top side. Preferably, the upper end of the passage channel ends in the base of the respective cavity, so that the base of the cavity is fluidically connected to the second side, in particular the underside of the substrate. This has the advantage that fluid can be discharged from the cavities via the through-channels without cells taken up in the cavities being carried along because of the smaller diameter of the through-channels compared to the diameter of the cells. Furthermore, the through-channels can be used to conduct a displacement medium into the cavities for removing the accommodated cells from the cavities. The displacement medium can be, in particular, a displacement fluid, i.e. a fluid in the form of a gas, for example nitrogen, a gas mixture, for example air, or a liquid, in particular a liquid having a very low surface tension, for example a fluorocarbon such as perfluorinated or partially fluorinated aliphatic hydrocarbon or an aliphatic chlorohydrocarbon or an aliphatic hydrocarbon.Depending on the position of the openings of the through-channels, the displacement medium can be introduced through the underside or a lateral side or side wall of the substrate into the substrate and thus into the cavities, preferably, as explained above, through the bottom of the cavities into the interior of the cavities. The microfluidic device can have a pump, in particular a membrane pump, and / or a pneumatic interface for introducing a displacement medium into the cavities, in particular via at least some of the recesses, preferably via the through channels.Preferably, at least some cavities have a plurality of such through-channels, wherein either all through-channels can lead to the underside or alternatively some through-channels can also lead to another side. The through channels can be at least partially fluidically connected to one another. In the case of the above-described poration, the through-channels can preferably be pore channels which can form in particular the above-described sponge-like structure in the poration substrate. For this purpose, the substrate can be completely porousized for the production of the pore channels. Alternatively, the partially, in particular layer-by-layer, porosified substrate can be abraded from a second side, in particular the underside, in the direction of the bases of the cavities until at least some of the through-channels fluidically connect at least some of the cavities to the underside. It should be noted that electrochemical poration generally ends at the moment the first pores open to the substrate back side, since from this moment there are short circuits between front and back side across the electrolyte. For this reason, a grinding process from the substrate back side is recommended in order to overcome the last nonporousized piece of substrate to the porousized material, or the use of an electroless poration method for the last piece.In a particularly preferred development, the substrate has a textile fabric, wherein the textile fabric is fluidically connected to recesses of at least some cavities, in particular via the corresponding passage channel, or comprises at least some of the recesses. The substrate can have the textile fabric in particular on the underside of the substrate. In particular, the textile fabric can thus adjoin the underside of the substrate or be arranged on the underside. A textile fabric can be understood in particular as an arrangement of a plurality of threads and / or fibers, in the following both also referred to as fibers, in particular comprising biogenic or mineral fibrous materials (natural fibers) occurring in nature and / or organic or inorganic fibrous materials (chemical fibers) created. The arrangement can have systematics, in particular a regular arrangement, at least in places or completely. However, the fibers can also be arranged at least partially with respect to one another without predefined regularity and in particular can be entangled or interwoven with one another. The textile fabric can alternatively or additionally have one or more (microporous) membranes, in particular comprising stretched (expanded) polytetrafluoroethylene (ePTFE), which are preferably waterproof as wet barrier membranes but are permeable to water vapor, such as the material of Gore-Tex® membranes. According to a special embodiment, the textile fabric can also be designed in the narrower sense as a fabric with fibers that in particular cross regularly with one another and, for example, have at least two fiber systems that cross one another at an angle in at least one plane of the textile fabric. The textile fabric may comprise fluorinated and / or non-fluorinated fibers, in particular synthetic fibers, for example in the form of the above-mentioned microporous membrane. The textile fabric can advantageously prevent undesired aqueous liquid from flowing out of the cavities through the recesses, but preferably displacement medium, for which the textile fabric is permeable, can be conveyed through the textile fabric via the recesses, in particular via the through-channels, into the cavities. In the case of at least partial poration of the substrate (described above), the textile fabric preferably adjoins at least partially a porous part of the substrate. According to a particular embodiment, the textile fabric forms the bottom of at least some of the cavities. In other words, in this embodiment, instead of a base of substrate material, a base of the textile fabric is provided, while the walls are preferably further formed by preferably porosified or nonporousized substrate material. The textile fabric can thus also completely replace the porousised substrate, in particular silicon, on the bottom of the cavities. In general, the invention thus also relates in a further aspect to a substrate for receiving and microfluidic analysis, in particular counting, of biological cells, in particular circulating tumor cells, wherein the substrate has cavities on an upper side for receiving the cells, wherein the substrate has a textile fabric, wherein the textile fabric is fluidically connected to at least some of the cavities and wherein the above-described recesses, in particular in the form of pores and / or through channels, are preferably developments of this second aspect.According to a particularly advantageous development of the invention, the textile fabric comprises a fluid, i.e. a gas and / or a liquid, for the heat transfer into the substrate and / or out of the substrate. This has the advantage that the associated increase in the thermal conductivity of the textile fabric together with the fluid absorbed in the textile fabric enables a more effective and faster temperature control of the substrate and in particular of the interior of the cavities. If the textile fabric comprises fluorinated fibers, in particular perfluorinated fabrics such as GoreTex® a gas, a gas mixture or preferably a fluorocarbon can be used as the fluid for heat transfer. If the textile fabric comprises non-fluorinated fibers, the fluid for heat transfer can comprise aliphatic hydrocarbons in particular. This fluid for the heat transfer can preferably also be used for the removal of the cells as displacement medium as described above. For this purpose, either even more such fluid can be introduced into the textile fabric, in particular pumped in, so that the fluid already present in the textile fabric is displaced into the cavities and in the process removes the contents of the cavities, in particular the cells, from the cavities. Alternatively or additionally, a second displacement medium can be introduced into the textile fabric, in particular pumped in, in order to press the fluid as the first displacement medium into the cavities.Brief Description of the DrawingsExemplary embodiments of the invention are schematically illustrated in the drawings and explained in more detail in the following description.They show FIG. 1 a shows a flow diagram of an exemplary embodiment of the method according to the invention for receiving and microfluidic analysis of biological cells, FIG. 1 bshows a flow diagram of an exemplary embodiment of the inventive method of producing an inventive substrate, FIG. 2 shows an exemplary embodiment of the device according to the invention, and FIGS. 3-8 show exemplary embodiments of the substrate according to the invention, preferably as part of the device according to the invention.Embodiments of the InventionFIG. 2 shows an exemplary embodiment of the microfluidic device 200 according to the invention comprising an exemplary embodiment of the substrate 100 according to the invention. The substrate 100 and / or the device 200 can preferably be based on a carrier substrate or microfluidic cartridge described in the laid-open specification DE 10 2021 203 897 A1. Substrate 100 and device 200 can be used, for example, for method 500 according to the present invention, for which an exemplary embodiment is described below with reference to the flow chart shown in FIG. 1 a.According to a first step 501 of the method 500, a blood sample, for example comprising 100-200 microliters (μl) of whole blood from a particularly immediately preceding blood collection in a human or animal, is provided and, in a second step, is input into an input chamber 201 of the device 200, which can subsequently preferably be closed. For example, for this sample, a number or concentration of predefined cells, in particular CTCs, is to be determined, also referred to below as target cells. Prior to introduction, a substance, for example plasmin, can be added to the sample for the purpose of unmasking the target cells, in particular CTCs, that is to say for the purpose of exposing structures detectable, for example via staining reagents, on the surface of the target cells.In the input chamber 201 or in another chamber 220 of the device 200, referred to below as treatment chamber 210, the sample can be mixed in a second step 502 with further liquids 80 or substances 80 which can be stored in a storage chamber 220 of the device 100. The liquids or substances are preferably substances for lysing erythrocytes, for example in the form of a commercially available erythrocyte lysis buffer (in particular ammonium chloride containing for minimal lysing action on nucleated cells). The sample with lysed erythrocytes obtained in this way is also referred to below as blood lysate. The substances 80 can also comprise colouring reagents as colour markers, in particular for CTCs, for example in the form of a colouring solution.The device 200 also comprises a chamber 230 (also referred to as substrate chamber 230 below) schematically illustrated in vertical cross section in FIG. 3 a, wherein the chamber 230 has a substrate 100 with cavities 120 on a surface 110 or upper side 110 of the substrate 100 for receiving and analyzing, in particular counting, biological cells. The substrate 100 may be disposed on the bottom 238 of the chamber 230 or recessed into the bottom 238, for example. FIG. 3 bshows a plan view of the chamber 230 and of the substrate 100 with, for example, circularly delimited cavities 120. The cavities 120 can preferably be formed as microcavities 120. The substrate 100 can be formed, for example, as a silicon array structured with microcavities, as described, for example, in DE 10 2021 203 897 A1. For example, the chamber has dimensions of 12.5 x 12.5 x 0.64 mm with a volume of 100 μL and substrate 100 dimensions of 13.45 x 13.45 x 0.38 mm. The cavities 120 have widths or diameters and depths in the one- to two-digit micrometer range, for example, for the respective recording of at least one cell, in particular a nucleated, preferably human cell, in particular CTC. The cavities are preferably designed to be able to accommodate a maximum of three cells of predefined size, preferably three cells mentioned above, in particular CTCs. As illustrated in FIGS. 3 aand 3 b, the cavities 120 can have a rectangular cross section or a circularly limited base area and thus each have the shape of a cylinder or alternatively of another sack-shaped depression 120. These microcavities 120 can be produced, as a first step 601 of the production method 600 according to the invention, in the silicon substrate using methods of semiconductor technology or microsystem technology, for example by plasma depth etching methods, in particular using the so-called "Bosch DRIE" process. Such poration processes are fundamentally known and have been used for many years, for example, in the case of Robert Bosch GmbH in large series for the production of piezoresistive surface microelectromechanical pressure sensors. In particular, porous silicon has a number of outstanding properties in addition to the permeability to gases and, to a limited extent, also to aqueous liquids; among others, it is highly biocompatible and prevents adhesion of biological cells on account of its nanostructure. The substrate chamber 230 has a first inlet 231 and a second inlet 232, wherein the two inlets 231, 232 are arranged in different walls of the chamber 230, in particular in opposite walls, so that the substrate 100 is preferably located between the two inlets 231, 232.In a third step 503, the blood lysate 10 is placed as a liquid phase above the surface of the substrate 100 in the so-called headspace 233, for example through the first inlet 231, so that at least some of the cells 40, in this example CTCs, can sediment into the cavities 120 of the substrate 100.FIG. 4 shows an enlarged illustration of a part of the substrate 100, wherein it is schematically illustrated that the bottom 121 and walls 122, 123 of the cavities 120 have recesses 130, which serve for reduced adhesion or adhesion of the accommodated cells 40 in the cavities 120. Preferably, the recesses 130, as shown, are smaller than the target cells 40 to be accommodated in the cavities 120.These recesses 130 can be formed, for example, in particular as pores, during the second step 602 of the production method 600 according to the invention. In this case, a layer 140 of the substrate can be porousised, wherein this layer comprises the cavities 120 at least partially and preferably completely (as illustrated in FIG. 4 ). If the substrate comprises silicon (Si for short) or consists of silicon, the surface and / or a zone of the substrate 100 close to the surface can be electrochemically converted into porous silicon preferably completely and over the whole area after production of the cavities 120. In this case, a certain Si layer thickness is converted by electrochemical anodization into a nanoporous or mesoporous Si layer, through which a plurality of nanoscale channels 130 pass and which forms a sponge-like silicon structure. In FIG. 4 and also FIGS. 5 to 7, the pores 130 are only drawn in a partial manner and are intended to illustrate that, depending on the etching process, the pores extend here mainly vertically from the upper side 110 to the lower side 150 with a certain degree of cross-linking. The process of electrochemical anodization or electrochemical etching of silicon in a hydrofluoric acid electrolyte, in particular an aqueous HF solution with addition of alcohols such as methanol, ethanol or isopropanol for reducing the surface tension, is well understood and can be carried out either electrolessly by addition of oxidizing agents to the hydrofluoric acid electrolyte (so-called HNA solutions: HNA=hydrofluoroic acid-HF, nitroic acid-HNO 3, acetic acid-CH 3 COOH) or by active electrical flow through the silicon wafers at wafer level. For this purpose, the silicon wafer is inserted into a reaction tank with hydrofluoric acid electrolytes between two electrodes-an anode and a cathode-with a seal on the wafer edge and contacted from both sides via the hydrofluoric acid electrolyte. The wafer front side (as a later surface 110 of the substrate 100) with the cavities 120 must be anodically poled with respect to the cathodic hydrofluoric acid volume lying in front. The anodic contacting of the wafer takes place via the anodic hydrofluoric acid volume which abuts the wafer underside by means of a p-doping of the wafer underside and / or an underside illumination. Both measures, p-doping and / or illumination of the underside, overcome the Schottky diode between hydrofluoric acid electrolyte and silicon wafer underside as a prerequisite for successful anodic contacting. Current density through the wafer and hydrofluoric acid concentration determine the degree of porosity, the process time determines the thickness of the porous layer. This technology has been established in MEMS fabrication fundamentally for many years and is used, for example, for mass-produced products such as surface microelectromechanical piezoresistive pressure sensors for consumer and automobile applications with numbers of more than 100 million per year.Since the porousized silicon layer is gas-permeable, the filling of the cavities takes place particularly without problems, since trapped gas in the cavities can easily be displaced through the porous layer. This effect helps to avoid gas bubbles enclosed in the cavities.The cells 40 collected in the cavities, which come to rest on the porous base 121, on which they come to rest and possibly also come into contact with the porous side walls 122, 123, do not build up any substantial bonds to the contact surfaces 121, 122, 123, i.e. do not stick on and therefore remain movable in the cavities 120. They can thus also be conveyed out of the cavities 120 again if necessary by the action of only small forces. This can be effected in a fifth step 505 of the method 500 after the sedimented cells 40 have been analyzed, in particular counted, in a fourth step 504. For the renewed removal 505 of the cells 40 from the cavities, for example, the force of gravity can be used again, which has caused the cells 40 to sink into the cavities 120 during the sedimentation phase 503. A change in position, in particular a tilting or turning over, of the device 200 can be used to transfer the cells 40 following the changed direction of the force of gravity out of the cavities 120, preferably into an aqueous phase adjoining the surface 110. The aqueous phase can then be moved, for example, into an analysis chamber 240 of the device 200 for further analysis or processing of the cells. Alternatively, the cells 40 can also be removed from the device 200, for example through an opening in the device 200, in particular a closable opening such as for a so-called LDT cartridge described in the laid-open specification DE 10 2021 211 545 A1. The opening can lead, for example, directly into the substrate chamber 230 or analysis chamber 240. For a well-defined removal, the aqueous phase can be taken off with a pipette or a syringe. This can be effected in particular depending on a previously carried out cell count 504 (for example a CTC count) in order to feed the cells 40 to a continuing analysis outside the device 200.In a further development of the invention, the electrochemical porosification of the substrate 100, in particular of the silicon substrate, can be advanced to such an extent that the porous layer reaches from the bottom 121 of the cavities 120 as far as the underside 150 of the substrate. For an economic process time and a well-defined process result, the substrate 100 is not completely porousized through, but rather only for a sufficiently long time to produce a porous layer of, for example, 100 to or a few 100 μm of porous silicon from the top side 11 0 in the direction of the bottom side 150. Thereafter, for example as a preferred third step 603 of the production method 600 according to the invention, the substrate 100 is mechanically ground or buffed back, i.e. rediluted from its underside 150 until the grinding process reaches the porous substrate layer. As schematically shown in FIG. 5, passages 124, in particular nanoscale passages 124 (for example a passage 124 shown in simplified form as a straight tunnel), are thus respectively achieved under the cavities 120 to the underside 150 of the substrate 100. This can be used advantageously to achieve particularly easy fillability of the cavities 120 with the aqueous sample medium, since gas inclusions in the cavities 120 can easily be displaced through the porous base toward the underside 150. The aqueous sample medium is substantially stopped at the porous substrate layer due to viscosity and surface tension. Alternatively or additionally to the formation of the vias 124 via poration of the substrate 100, in particular straight vias 124 may also be formed via anisotropic etching, in particular using the "Bosch DRIE" process.Alternatively or additionally to the change in position described above, in the fifth step 505, a displacement medium, in particular a fluid, can also be used for removing the cells 40, in particular together with liquid located in the cavities, from the cavities 120. The fluid can be a gas, for example nitrogen, or a gas mixture, for example air, or a liquid, for example a liquid with low surface tension, such as fluorocarbon (perfluorinated aliphatic hydrocarbon) or else chlorohydrocarbon or hydrocarbon, such as n-hexane or n-heptane. For this purpose, the displacement medium can be conducted over the surface 110 of the substrate 100 for flushing out the cavities 120. In the case of passages 124 to the cavities 120, the fluid may be advantageously directed through these passages 124 to remove the cells 40 from the cavities. For this purpose, as schematically shown in FIG. 2, the device 200 can include a pump 250, for example a diaphragm pump customary in microfluidics, and / or a pneumatic interface 250 via introduction of the displacement medium into the cavities 120, in particular via at least some of the recesses 130 as through-channels. The pump 250 or interface 250 can be connected directly to the substrate 100, in particular to the underside of the substrate 100, via a channel 234, as also indicated in FIG. 3 a.In both exemplary embodiments according to FIGS. 4 and 5, the substrate 100, in particular the silicon substrate, can be ground ("background") from the underside 150 in the direction of the upper side 110, before and / or after the poration in order to produce the recesses 130. In FIGS. 4 and 5, it is shown by way of example by means of the dashed line 300 that this back-grinding is preferably carried out to such an extent that the layer 140 with completely porousised substrate material or a porous substrate layer of reduced thickness remains below the cavities 120. I.FIGS. 6 and 7 show exemplary embodiments of an advantageous development of the invention, in which a textile fabric 160 is applied, for example adhesively bonded in places, to the underside 150, preferably over the whole area. For example, the textile fabric 160 can be adhered along an edge to the underside 150 of the substrate, for example via an adhesive frame, so that there is a fluidic connection between the textile fabric and the cavities 120, in particular via the pores and / or through-channels 124. The textile fabric 160 may comprise, for example, GoreTex® and / or another fabric with fluorinated and / or non-fluorinated synthetic fibers. Between the bottom 121 of the cavities 120 and the textile fabric 160 there may be still a porousised or passaged substrate layer as shown in Fig. 6. Alternatively, the fabric 160 can directly adjoin the cavities 120 and thus form the base 121 of the cavities 120, as shown in FIG. 7. For example, the textile fabric can have a thickness of 100-500 μm. In these embodiments, too, there is the particular advantage of the fact that the cavities 120 can be easily filled with an aqueous sample medium, in that gas bubbles in the cavities 120 can easily pass, if appropriate, through the porous substrate residual layer in the direction of the underside 150 and the textile fabric 160 until the aqueous sample liquid arrives there and can practically pass through it not at all (at GoreTex®) or only poorly (at non-fluorinated synthetic fiber fabric). Cells 40 which are sedimented into the cavities 120 do not fundamentally stick to perfluorinated tissue (GoreTex®). When using non-fluorinated synthetic fibre fabric, a strength of suppression of adhesion of the cells depends on the specific choice in this group of fibres - for example plastics with only a few polar groups can be chosen, for example polystyrene (foam), polyethylene or polypropylene. In the preferred case of Fig. 6, adhesion of the cells 40 through the porous substrate residual layer is practically avoided.The textile fabric 160 can also have a fluid 161 for the heat transfer into the substrate 100 and / or out of the substrate 100 (as indicated in FIG. 6 ) in order to conduct heat more effectively and quickly in particular into the cavities 120 or out of the cavities 120 and thus to bring about a temperature change in the cavities 120. When using fluorinated fibers in the textile fabric 160, particularly in the case of GoreTex® the fluid 161 may preferably contain fluorocarbon. Fluorocarbon materials are excellent thermal conductors and are readily absorbed by (per)fluorinated tissue. When non-fluorinated fibers are used in the fabric 160, the fluid 161 may preferably comprise aliphatic hydrocarbons.For the removal of the cells 40 in the fifth step 505, as described above, a displacement medium can be used which is pressed through the textile fabric 160 in the direction of the cavities 120. When using GoreTex® or other perfluorinated fabrics, a gas, a gas mixture or a fluorocarbon is preferred, while when using non-fluorinated synthetic fiber fabric, a low viscosity oil or aliphatic hydrocarbon with low surface tension is advantageous. Preferably, the above-described fluid already located in the textile fabric 160 for the heat transfer 161 can also be used as a displacement medium, for example by introducing further such fluid 161 or another displacement medium into the textile fabric 160 and thus pressing the fluid 161 located there into the cavities 120.The use of porous substrate, in particular silicon, can thus be effected both alternatively to or additionally together with a synthetic fiber fabric termination of the substrate bottom side. The advantages of both solutions can thus be combined in many ways. According to a further embodiment, it is also possible to completely dispense with the porosification of the substrate 100 and to use only the synthetic fiber fabric termination of the cavities 120, as shown in FIG. 8, wherein in this variant a certain risk of the cells adhering to the side walls 122, 123 of the cavities 120 remains. In all cases, the permeability of the bottom of the wells to gases or liquids of low viscosity and surface tension allows active and gentle later displacement of the sample medium with the cells 40 contained therein to make them accessible for removal.

Claims

Substrate (100) for receiving and microfluidic analysis, in particular counting, of biological cells (40), in particular circulating tumour cells (40), wherein the substrate (100) has cavities (120) on an upper side (110) for receiving the cells (40), characterized in that walls (121, 122) and / or bases (123) of at least some cavities (120) have recesses (130) (130), in particular pores, for supporting removal of received cells (40) from the cavities (120).The substrate (100) according to claim 1, wherein at least some of the cavities (120) have more than 10, preferably more than 100, most preferably more than 500 recesses (130)Substrate (100) according to one of the preceding claims, wherein a part, in particular a layer (140), of the substrate (100) comprising at least some of the cavities (120) is porousised, in particular meso- and / or nano-porousised, and wherein at least some of the recesses (130) are formed as pores.Substrate (100) according to one of the preceding claims, wherein at least one recess (130), preferably at least one recess (130) in a plurality of cavities (120) in each case, extends as a through channel (124) as far as a second side (150) of the substrate (100), in particular an underside (150) of the substrate (100) opposite the top side (121).Substrate (100) according to one of the preceding claims, wherein the substrate (100) has, in particular on the underside (150) of the substrate (100), a textile fabric (160), in particular comprising fluorinated and / or non-fluorinated fibers, wherein the textile fabric (160) is fluidically connected to recesses (130) of at least some cavities (120), in particular via the through channel (124) or comprises at least some of the recesses (130).The substrate (100) of claim 5, wherein the textile fabric (160) forms a bottom (121) of at least some of the cavities (120).The substrate (100) of claim 5 or 6, wherein the textile fabric (160) comprises a fluid (161) for heat transfer.Microfluidic device (200), in particular microfluidic cartridge, comprising a substrate (100) according to one of the preceding claims.Microfluidic device (200) according to claim 8, comprising a pump (250) and / or a pneumatic interface (250) for introducing a displacement medium into the cavities (120), in particular via at least some of the recesses (130), for removing cells (40) accommodated in the cavities (120).Method (500) for recording and microfluidic analysis, in particular counting, of biological cells (40), in particular circulating tumour cells (40), having a substrate (100) according to one of Claims 1 to 7 or a device (200) according to one of Claims 8 or 9, comprising the steps: • recording cells (40) in at least some of the cavities (120), in particular via sedimentation of the cells (40) from a sample. • Preferably analysis, in particular counting, of the recorded cells (40). • Removal of at least some of the recorded cells (40) from the cavities (120)Method (500) according to claim 10, wherein the removal takes place with the aid of gravity, in particular by tilting, turning or inverting the substrate (100) in the gravitational field.Method (500) according to claim 10 or 11, wherein the removal of the cells (40) is or is carried out by introducing a displacement medium, in particular a gas or a liquid, in particular a fluid (161) for heat transfer situated in the textile fabric (160), through the recesses (130) into the cavities (120), wherein preferably the displacement medium is introduced through a side wall and / or through the underside (150) of the substrate (100) and is preferably moved through the base (121) of the cavities (120) into the cavities (120).Method (600) for producing a substrate (100) according to any one of claims 1 to 7, comprising the steps • producing (601) the substrate (100) with the cavities (120) on the surface (110), • forming (602) the recesses (130) in the cavities (120), in particular by porosifying at least a part of the substrate (100)Method (600) according to claim 13; wherein the substrate (100) is abraded from a second side, in particular the underside, in the direction of the bases (123) of the cavities (120) until at least some of the recesses (130) (130), as through-channels (124), fluidically connect at least some cavities (120) to the underside.Microfluidic device (200), in particular according to claim 8 or 9, configured to perform a method (500) according to any of claims 9 to 12.

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