Cavity substrate with textile fabric for receiving and microfluidically analysing, in particular counting, biological cells, in particular circulating tumour cells

Abstract

The invention relates to a substrate (100) for receiving and microfluidically analysing, in particular counting, biological cells (40), in particular circulating tumour cells (40), wherein the substrate (100) comprises, on a top side (110), cavities (120) for receiving the cells (40), characterised in that the substrate (100) comprises a textile fabric (160), wherein the textile fabric (160) is fluidically connected to at least some of the cavities (120). The invention also relates to a microfluidic device (200), to a method (500) for receiving and microfluidically analysing biological cells (40) using a substrate (100) of this kind, and to a method (600) for producing a substrate (100) of this kind.

Description

[0001] Cavity substrate with textile fabric for the capture and microfluidic analysis, in particular counting, of biological cells, especially circulating tumor cells

[0002] State of the art

[0003] Circulating tumor cells (CTCs) are cells that can detach from the primary tumor, penetrate the peripheral bloodstream, and then reach surrounding tissue. These cells, which are present in the blood even despite surgery and therapy, are referred to as “minimum residual disease (MRD)” and are considered 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 (CTC count) is relevant for both prognostic statements and clinical management (Cristofanilli et al., Circulating tumor cells, disease progression, and survival in metastatic breast cancer. Semin. Oncol. 33, 2006, 3, p.9-14), particularly for the detection of metastasis at an early stage and for efficiency control and adjustment during a therapy procedure.

[0004] The quantification of CTCs from a so-called "liquid biopsy," a blood sample from a tumor patient, has the advantage that it can be performed as often as desired, as it is a minimally invasive procedure involving a blood draw. The greatest challenge is the very small number of CTCs relative to blood cells—especially in the MRD stage or in the very early stages of metastasis. CTC detection therefore requires highly sensitive detection methods to detect the very small number of CTCs among millions of red and white blood cells early and efficiently.

[0005] A sedimentation array offers a method for quantifying CTCs from a blood sample after fluorescent staining with tumor cell-specific antibodies of one type (e.g., EpCAM-FITC) or of multiple types (e.g., EpCAM, cytokeratin, HER2-neu, etc.) using optical detection with as little loss as possible. This allows nucleated cells from a blood sample to settle in wells of a single plane after erythrocyte lysis, where they can be detected and counted with spatial resolution using optical systems. Furthermore, the capture of the cells in these wells allows for flushing with various media and reagents to remove excess lysis buffer, erythrocyte lysate, free dyes, or similar substances, and / or to add additional dye labels without the target cells changing their position or being flushed out again.The published patent application DE 10 2021 203 897 A1 describes a suitable microfluidic cartridge comprising a carrier substrate with a plurality of etched depressions as a sedimentation array, into which CTCs can be sedimented and subsequently analyzed and counted by means of their various fluorescent labels.

[0006] Disclosure of the invention

[0007] Advantages of the invention

[0008] Against this background, the invention relates 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.

[0009] To absorb the cells, a sample containing the cells, in particular blood, can preferably be applied to the upper side of the substrate, preferably after pretreatment of the blood, for example lysis of blood components, in particular erythrocytes, and / or filtering of certain blood particles. The cells from the sample can sediment into at least some of the wells. The blood can be whole blood. The sample can have been obtained by a blood sample taken from a human or animal, in particular immediately beforehand. 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 above, can be used and referred to as a sedimentation array and is based on the sedimentation array described in the published patent application DE 10 2021 203 897 A1 (referred to therein as a carrier substrate), is preferably silicon or a material comprising silicon. Alternatively, the substrate can comprise, for example, germanium, aluminum, titanium, tungsten, or consist of one of these materials. Cavities are to be understood in particular as sack-shaped depressions on the upper side of the substrate. The substrate can be arranged in a microfluidic device for analyzing such cells. The invention thus also relates to a microfluidic device, in particular a microfluidic cartridge, comprising a substrate according to the invention.Furthermore, the invention relates to a method for producing such a substrate, wherein, in addition to producing the cavities, which can preferably be carried out using an anisotropic etching process, particularly in the case of silicon, the textile fabric is arranged on the substrate, in particular on an underside of the substrate opposite the top side, whereby the textile fabric is fluidically connected to at least some of the cavities. According to a preferred embodiment, the substrate is ground from a second side, in particular the underside, towards the bottoms of the cavities to such an extent that, when the textile fabric is arranged on the second side, at least some of the cavities are fluidically connected to the textile fabric.

[0010] A textile fabric can be understood in particular as an arrangement of several threads and / or fibers, hereinafter both also referred to as fibers, in particular comprising naturally occurring biogenic or mineral fibers (natural fibers) and / or created organic or inorganic fibers (man-made fibers). The arrangement can have a systematic, in particular a regular arrangement, at least in part or entirely. However, at least in part, the fibers can also be arranged relative to one another without any predetermined regularity and, in particular, can be entangled or woven with one another. Alternatively or additionally, the textile fabric can have one or more (micro)porous membranes, in particular comprising stretched (expanded) polytetrafluoroethylene (ePTFE), which are preferably waterproof as moisture barrier membranes but 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, in particular, regularly intersecting fibers and, for example, have at least two fiber systems intersecting with each other at an angle in at least one plane of the textile fabric. The textile fabric can comprise fluorinated and / or non-fluorinated fibers, in particular synthetic fibers, for example in the form of the aforementioned microporous membrane. The textile fabric can advantageously prevent undesired aqueous liquid from flowing out of the cavities, but preferably displacer medium, for which the textile fabric is permeable, can be transported into the cavities via the textile fabric. The substrate can have the textile fabric, in particular on the underside of the substrate. In particular, the textile fabric can thus border the underside of the substrate or be arranged on the underside.

[0011] A fluidic connection of the textile fabric with at least some of the cavities is understood in particular to mean that fluids permeable to the textile fabric can pass from the textile fabric into these cavities and vice versa. The fluidic connection can be realized in particular by channels between these cavities and the textile fabric. In particular, the channels can be formed via anisotropic etching or drilling, depending on the substrate material. Thus, these cavities are fluidically connected via the channels thus formed as through-channels to a second side of the substrate, in particular to an underside opposite the top side of the substrate, and thus preferably to the textile fabric adjacent to the second side.

[0012] Alternatively, the channels or through-channels can be formed as pores through at least partial porosification of the substrate. Porosification of the substrate is understood to mean, in particular, an etching process, in particular an electrochemical etching process in electrolytes containing hydrofluoric acid or, alternatively, an electroless etching process in electrolytes containing hydrofluoric acid in conjunction with oxidizing agents (for example, nitric acid combined with acetic acid, for example as HNA = hydrofluoric acid (H), nitric acid (N), and acetic acid (A)), whereby a substrate region, in particular a silicon region, is converted into a porous, sponge-like material in a controlled manner. The thickness of the surface layer thus porosified can be largely freely selected over the process time and is well-defined in a time-controlled manner.In the case of such at least partial porosification of the substrate, the textile fabric preferably borders at least partially on a porous part of the substrate. As an alternative to the porosification of silicon, the substrate to be porosified can, as described above, comprise, for example, germanium, aluminum, titanium, tungsten, or consist of one of these materials.

[0013] In an advantageous development, a part, in particular a layer, of the substrate comprising at least some of the cavities is porous, in particular meso- and / or nanoporous. This has the advantage that the floor and walls of these cavities have pore openings, which significantly reduce the risk of adhesion of the cells accommodated in the cavities. In the case of porous silicon and similarly acting materials, the high biocompatibility is advantageously utilized, whereby the porosity significantly hinders the growth or adhesion of biological substances, in particular cells or proteins, and thus "clogging" or "biofouling" is avoided.The cells accommodated in the cavities, which come into contact with the porous (silicon) base on which they rest, and possibly also with the porous (silicon) sidewalls, do not form bonds with the contact surfaces, i.e., they do not stick, but remain mobile in the recesses. They can therefore advantageously be expelled from the cavities when needed by the application of only small forces. As an alternative to pores, the walls and / or floors of at least some of the cavities can also have recesses produced by other processes, in particular by etching or drilling, which make it difficult for the cells to adhere to the walls or floors and thus facilitate later removal from the cavities.

[0014] The substrate can also be fully porosified to create the pore channels. Alternatively, the partially porosified substrate, especially layer-by-layer, can be ground from a second side, especially the underside, toward the bottom of the cavities until at least some of the through-channels fluidically connect at least some of the cavities with the underside. It should be noted that electrochemical porosification generally ends the moment the first pores break through to the back of the substrate, since from this moment on, short circuits exist between the front and back sides via the electrolyte. For this reason, a grinding process from the back of the substrate is recommended to overcome the last non-porosified piece of substrate to the porosified material, or the use of an electroless porosification process for the last piece.

[0015] The pores are, in particular, smaller than the size of the cells to be captured and analyzed, preferably smaller than typical circulating tumor cells, especially smaller than epithelial cells. For example, the pores have a size between 1 nm and 100 nm (corresponding to nanoporosity), a size of 0.1 - 1 pm (corresponding to mesoporosity), or a size of 1 - 5 pm (corresponding to macroporosity). The size of the pores can be determined, in particular, by the etching process (current density, electrolyte concentration, oxidizing agent concentration, doping level in the silicon). The recesses described above can also have a size within these size ranges.

[0016] Some of the through-channels can also be directed to surfaces or sides of the substrate that are not adjacent to the textile fabric, as required. The through-channels, in particular pores, can be used to conduct a displacement medium as mentioned above into the cavities to remove the captured cells from the cavities. The displacement medium can, in particular, be 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 with very low surface tension, for example, a fluorocarbon such as a perfluorinated or partially fluorinated aliphatic hydrocarbon, or an aliphatic chlorohydrocarbon, or an aliphatic hydrocarbon.The displacement fluid can preferably be conveyed into the cavities via the textile fabric and the through-channels opening into the textile fabric, if the fluid is permeable to the textile fabric. If the textile fabric comprises fluorinated fibers, in particular perfluorinated fabric as in GoreTex®, a gas, a gas mixture or preferably a fluorocarbon can be used as the displacement fluid. If the textile fabric comprises non-fluorinated fibers, the displacement fluid can in particular comprise or consist of aliphatic hydrocarbons. Depending on the position of the openings of the through-channels or the position of the textile fabric, the displacement medium can be introduced into the substrate and thus into the cavities through the underside or a lateral side or side wall of the substrate, preferably through the bottom of the cavities into the interior of the cavities.The microfluidic device may comprise a pump, in particular a diaphragm pump, and / or a pneumatic interface for introducing a displacement medium into the cavities, in particular via the textile fabric or via the through-channels.

[0017] According to a particular embodiment of the invention, the textile fabric forms the bottom of at least some of the cavities, so that there is a direct fluidic connection between these cavities and the textile fabric. In other words, in this embodiment, instead of a bottom made of substrate material, a bottom made of the textile fabric is provided, while the walls are preferably still formed by preferably porous or non-porous substrate material.

[0018] According to a particularly advantageous development, the textile fabric comprises a fluid, i.e., a gas and / or a liquid, for heat transfer into and / or out of the substrate. This has the advantage that the resulting increase in the thermal conductivity of the textile fabric, including the fluid absorbed in the textile fabric, enables more effective and faster temperature control of the substrate and, in particular, the interior of the cavities. The fluid for heat transfer can be a fluid such as that described above for the displacement medium. This fluid for heat transfer contained in the textile fabric can then also advantageously be used as a displacement medium for emptying the cavities.

[0019] The invention also relates to a method for analyzing, in particular counting, biological cells, in particular circulating tumor cells, using such a substrate, wherein at least some of the cells preferably taken up into the cavities via sedimentation are removed from the cavities again after a preferential analysis, in particular counting.

[0020] In the simplest embodiment of the method according to the invention, gravity is used again, as before in the preferential sedimentation. The gravity, which caused the cells to sink into the cavities during the preferential sedimentation phase, is used by tilting, turning, or inverting the substrate or the entire microfluidic device or cartridge including the substrate to transport the cells out of the cavities following the changed direction of gravity. In particular, the cells can be transferred into an aqueous phase initially above the surface of the substrate, where, after a certain waiting time, they can be removed with a pipette or syringe or transported by flow to an exit (and a removal station located there).The removal can be carried out in particular after completion of a count of the collected cells, for example in order to subject the cells to further analysis outside the cavities depending on the counting results.

[0021] Alternatively or additionally, the removal of the cells can be supported or carried out by introducing the above-described displacement medium, in particular via the textile fabric, and / or the fluid in the textile fabric for heat transfer into the cavities.

[0022] Short description of the drawings

[0023] Embodiments of the invention are illustrated schematically in the drawings and explained in more detail in the following description.

[0024] It shows

[0025] Figure 1 a is a flow diagram of an embodiment of the method according to the invention for recording and microfluidic analysis of biological cells,

[0026] Figure 1 b is a flow diagram of an embodiment of the inventive manufacturing method of a substrate according to the invention,

[0027] Figure 2 shows an embodiment of the device according to the invention and

[0028] Figures 3-6 show exemplary embodiments of the substrate according to the invention, preferably as part of the device according to the invention. Embodiments of the invention

[0029] Figure 2 shows an embodiment of the microfluidic device 200 according to the invention, comprising an embodiment of the substrate 100 according to the invention. Preferably, the substrate 100 and / or the device 200 can be based on a carrier substrate or microfluidic cartridge described in published patent application DE 10 2021 203 897 A1. Substrate 100 and device 200 can be used, for example, for the method 500 according to the invention, an embodiment of which is described below with reference to the flow diagram shown in Figure 1a.

[0030] 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 draw from a human or animal, is provided and, in a second step, introduced into an input chamber 201 of the device 200, which can then preferably be closed. For example, a number or concentration of predetermined cells, in particular CTCs, hereinafter also referred to as target cells, is to be determined for this sample. Before introduction, a substance, for example plasmin, can be added to the sample to unmask the target cells, in particular CTCs, i.e., to expose structures on the surface of the target cells that can be detected, for example, using staining reagents.

[0031] In the input chamber 201 or in another chamber 220 of the device 200, hereinafter referred to as the processing 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 pre-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 containing ammonium chloride for minimal lysis effect on nucleated cells). The sample thus obtained with lysed erythrocytes is hereinafter also referred to as blood lysate. The substances 80 can also comprise staining reagents as color markers, in particular for CTCs, for example in the form of a staining solution.

[0032] The device 200 also comprises a chamber 230 (hereinafter also referred to as substrate chamber 230), shown schematically in vertical cross-section in Figure 3a, wherein the chamber 230 has a substrate 100 with cavities 120 on a surface 110 or top side 110 of the substrate 100 for receiving and analyzing, in particular counting, biological cells. The substrate 100 can, for example, be arranged on the bottom 238 of the chamber 230 or embedded in the bottom 238. Figure 3b shows a plan view of the chamber 230 and of the substrate 100 with, for example, circularly defined cavities 120. The cavities 120 can preferably be shaped as microcavities 120. The substrate 100 can, for example, be designed 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 pL and substrate 100 dimensions of 13.45 x 13.45 x 0.38 mm. The cavities 120 have, for example, widths or diameters and depths in the single- to double-digit micrometer range for each accommodating 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 a predetermined size, preferably three of the aforementioned cells, in particular CTCs. As shown in Figures 3a and 3b, the cavities 120 can have a rectangular cross-section or a circularly delimited base area and thus each have the shape of a cylinder or, alternatively, another sack-shaped depression 120. These microcavities 120 can, as a first step 601 of the manufacturing method 600 according to the invention, be produced using methods of semiconductor technology orMicrosystems technology can produce porosity in the silicon substrate, for example, through deep plasma etching processes, particularly using the so-called "Bosch DRIE" process. Such porosification processes are generally known and have been used for many years, for example, by Robert Bosch GmbH in large-scale production of piezoresistive surface micromechanical pressure sensors. Porous silicon, in particular, has a number of outstanding properties besides its permeability to gases and, to a limited extent, to aqueous liquids. Among other things, it is highly biocompatible and, due to its nanostructure, prevents the adhesion of biological cells.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.

[0033] 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.

[0034] Figures 4 and 5 show an enlarged view of a portion of the substrate 100 according to two exemplary embodiments, schematically illustrating that a textile fabric 160 is applied, for example, glued in places, to the underside 150, preferably over the entire surface. For example, the textile fabric 160 can be glued along an edge to the underside 150 of the substrate, for example via an adhesive frame, so that a fluidic connection exists between the textile fabric and the cavities 120, in particular via the pores and / or through-channels 124. The textile fabric 160 can 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, as shown in Figure 4, there can also be a substrate layer that is porous or provided with passages by other means, in particular anisotropic etching.Alternatively, the fabric 160 can directly border the cavities 120 and thus form the bottom 121 of the cavities 120, as shown in Figure 5. For example, the textile fabric can have a thickness of 100-500 μm. The fabric 160, in the example according to Figure 4 in interaction with the pores of the substrate 100, advantageously enables easier filling of the cavities 120 with an aqueous sample medium, in that gas bubbles in the cavities 120 can easily pass through the porous substrate residual layer towards the underside 150 and the textile fabric 160 until the aqueous sample liquid reaches there and can penetrate practically not at all (with GoreTex®) or only with difficulty (with non-fluorinated synthetic fiber fabric). Cells 40 that are sedimented into the cavities 120 generally do not adhere to perfluorinated fabric (GoreTex®).When using non-fluorinated synthetic fiber fabric, the degree of suppression of cell adhesion depends on the specific material selected within this group. For example, plastics with only a few polar groups can be chosen, such as polystyrene (foam), polyethylene, or polypropylene. In the advantageous case shown in Figure 4, adhesion of the cells 40 is practically prevented by the porous residual substrate layer.

[0035] The textile fabric 160 may also include a fluid 161 for heat transfer into the substrate 100 and / or out of the substrate 100 (as indicated in Figure 4) in order to conduct heat more effectively and quickly, in particular into the cavities 120 or out of the cavities 120, and thus cause 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 fluorocarbons. Fluorocarbons are excellent heat conductors and are readily absorbed by (per)fluorinated fabric. When using non-fluorinated fibers in the textile fabric 160, the fluid 161 may preferably comprise aliphatic hydrocarbons.

[0036] As also schematically illustrated in Figures 4 and 5, the bottom 121 and / or the walls 122, 123 of the cavities 120 can have recesses 130, which support reduced adhesion or sticking of the accommodated cells 40 in the cavities 120. Preferably, as illustrated, the recesses 130 are smaller than the target cells 40 to be accommodated in the cavities 120. These recesses 130 can, in particular, be formed as the aforementioned pores during the second step 602 of the manufacturing method 600 according to the invention. In this case, a layer 140 of the substrate can be porous, wherein this layer at least partially and preferably completely encompasses the cavities 120 (as shown in Figure 4, for example, extending from the top side 110 to the dashed line 300) and preferably the pores or other through-channels 124 for the fluidic connection extend to the textile fabric 160.If the substrate comprises silicon (Si for short) or consists of silicon, the surface and / or a near-surface zone of the substrate 100 can be electrochemically converted into porous silicon, preferably completely and over the entire surface, after the cavities 120 have been formed. In this case, a certain Si layer thickness is converted by electrochemical anodization into a nanoporous or mesoporous Si layer, which is traversed by a multitude of nanoscale channels 130 and forms a sponge-like silicon structure.The process of electrochemical anodization or electrochemical etching of silicon in a hydrofluoric acid electrolyte, particularly an aqueous HF solution with the addition of alcohols such as methanol, ethanol, or isopropanol to reduce surface tension, is well-understood and can be performed either electrolessly by adding oxidizing agents to the hydrofluoric acid electrolyte (so-called HNA solutions: HNA = Hydrofluoric Acid - HF, Nitric Acid - HNO3, Acetic Acid - CH3COOH) or by actively applying electrical current through the silicon wafer at the wafer level. For this purpose, the silicon wafer is placed in a reaction tank containing hydrofluoric acid electrolyte between two electrodes – an anode and a cathode – with a seal at the wafer edge and contacted from both sides via the hydrofluoric acid electrolyte. The wafer front side (as the later surface 110 of the substrate 100) with the cavities 120 must be anodically poled relative to the cathodic hydrofluoric acid volume in front of it.The anodic contacting of the wafer is achieved via the anodic hydrofluoric acid volume applied to the wafer's underside by means of p-doping of the wafer underside and / or underside illumination. Both measures, p-doping and / or underside illumination, overcome the Schottky diode between the hydrofluoric acid electrolyte and the silicon wafer underside, which is a prerequisite for successful anodic contacting. Current density through the wafer and hydrofluoric acid concentration determine the degree of porosity, while the process time determines the thickness of the porous layer. This technology has been established in MEMS manufacturing for many years and is used, for example, for large-scale products such as surface micromechanical piezoresistive pressure sensors for consumer and automotive applications, with quantities exceeding 100 million per year.Because the porous silicon layer is gas-permeable, filling the cavities is particularly easy, as trapped gas can be easily displaced through the porous layer. This effect helps prevent gas bubbles from becoming trapped in the cavities.

[0037] The cells 40 collected in the cavities, which come into contact with the porous base 121 or with the base 121 made of textile fabric 160 on which they rest, and possibly also with the preferably porous side walls 122, 123, do not form substantial bonds to these contact surfaces 121, 122, 123, thus do not stick and therefore remain mobile in the cavities 120. They can thus be transported out of the cavities 120 again if necessary, even by the application of only small forces. This can take place 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, gravity can be used again, which caused the cells 40 to sink into the cavities 120 during the sedimentation phase 503.A change in position, in particular tilting or turning, of the device 200 can be used to transfer the cells 40 out of the cavities 120, preferably into an aqueous phase adjacent to the surface 110, following the changed direction of gravity. 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 published patent application DE 10 2021 211 545 A1. The opening can, for example, lead directly into the substrate chamber 230 or analysis chamber 240. For a well-defined removal, the aqueous phase can be tapped off with a pipette or syringe.This can be done in particular depending on a previously carried out cell count 504 (for example a CTC count) in order to supply the cells 40 for further 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 extends from the bottom 121 of the cavities 120 to the underside 150 of the substrate. For an economical process time and a well-defined process result, the substrate 100 is not completely porosified, but only over a sufficiently long time to produce a porous layer of, for example, 100 to or a few 100 pm of porous silicon from the top side 110 towards the underside 150. Thereafter, for example as a preferred third step 603 of the manufacturing method 600 according to the invention, the substrate 100 is mechanically ground orground back, i.e. thinned back from its underside 150, until the grinding process reaches the porous substrate layer. As also schematically shown in Figure 4, in particular nanoscale pore passages 124 (for example, a passage 124 is shown simplified as a straight tunnel) are thus reached under the cavities 120 to the underside 150 of the substrate 100. This can advantageously be used to achieve particularly easy filling of the cavities 120 with the aqueous sample medium, since gas inclusions in the cavities 120 can easily be displaced through the porous base to the underside 150. The aqueous sample medium is practically stopped at the porous substrate layer due to viscosity and surface tension.Alternatively or in addition to producing the passages 124 by porosification of the substrate 100, in particular straight passages 124 can also be produced by anisotropic etching, in particular using the “Bosch-DRIE” process.

[0038] According to a further embodiment, it is also possible to dispense entirely with the porosification of the substrate 100 and to use only the synthetic fiber fabric closure of the cavities 120, as shown in Figure 6, although in this variant, a certain risk of the cells sticking to the side walls 122, 123 of the cavities 120 remains. In all cases, the permeability of the bottom of the recesses to gases or to liquids with low viscosity and surface tension enables an active and gentle subsequent displacement of the sample medium with the cells 40 contained therein in order to make them accessible for removal. Alternatively or in addition to the change in position described above, a displacement medium, in particular a fluid, can also be used in the fifth step 505 to remove the cells 40, in particular together with liquid present in the cavities, from the cavities 120.The fluid can be a gas, such as nitrogen, or a gas mixture, such as air, or a liquid, such as a liquid with low surface tension such as fluorocarbon (perfluorinated aliphatic hydrocarbon) or a chlorohydrocarbon or hydrocarbon such as n-hexane or n-heptane. For this purpose, the displacement medium can be passed over the surface 110 of the substrate 100 to flush the cavities 120. In the case of passages 124 to the cavities 120, the fluid can advantageously be passed through these passages 124 to remove the cells 40 from the cavities.For this purpose, as schematically illustrated in Figure 2, the device 200 can comprise a pump 250, for example, a diaphragm pump commonly used in microfluidics, and / or a pneumatic interface 250 for introducing the displacement medium into the cavities 120, in particular via the textile fabric 106 and, depending on the design of the substrate 100, 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 Figure 3a.

Claims

Claims 1 . Substrate (100) for receiving and microfluidic analysis, in particular counting, of biological cells (40), in particular circulating tumor cells (40), wherein the substrate (100) has cavities (120) on a top side (110) for receiving the cells (40), characterized in that the substrate (100) is a textile fabric (160), wherein the textile fabric (160) is provided with at least some of the cavities (120) is fluidly connected.

2. Substrate (100) according to claim 1, wherein the textile fabric (160) has a bottom (121) of at least some of the cavities (120).

3. Substrate (100) according to claim 1 or 2, wherein the textile fabric (160) comprises a fluid (161) for heat transfer.

4. Substrate (100) according to one of the preceding claims, wherein walls (121, 122) and / or bottoms (123) of at least some cavities (120) have recesses (130), in particular pores, to assist in the removal of accommodated cells (40) from the cavities (120).

5. Substrate (100) according to claim 4, wherein the textile fabric comprises fluorinated and / or non-fluorinated fibers.

6. Substrate (100) according to one of the preceding claims, wherein at least one cavity (120) is fluidically connected via a through-channel (124) to a second side (150) of the substrate (100), in particular to a bottom side (150) of the substrate (100) opposite the top side (121), and preferably to the textile fabric (160).

7. 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) are porous, in particular meso- and / or nanoporous.

8. Microfluidic device (200), in particular microfluidic cartridge, comprising a substrate (100) according to one of the preceding claims.

9. 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 the textile fabric (160), for removing cells (40) accommodated in the cavities (120).

10. A method (500) for the recording and microfluidic analysis, in particular counting, of biological cells (40), in particular circulating tumor cells (40), with 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: • Intake of cells (40) into 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 cells taken (40). • Removing at least some of the captured cells (40) from the cavities (120) 11. Method (500) according to claim 10, wherein the removal is carried out with the aid of gravity, in particular by tilting, turning or inverting the substrate (100) in the gravitational field.

12. The method (500) according to claim 10 or 11, wherein the removal of the cells (40) is assisted or carried out by introducing a displacement medium, in particular a gas or a liquid, via the textile fabric (160) and / or via through-channels (124) into the cavities (120), in particular comprising conveying a fluid (161) located in the textile fabric (160) into the cavities for heat transfer.

13. A 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). • Arranging (602) a textile fabric (160) on the substrate (100), in particular on a bottom side (150) of the substrate (100) opposite the top side (110), whereby the textile fabric (160) is fluidically connected to at least some of the cavities (120).

14. The method (600) according to claim 13, wherein the substrate (100) is ground from a second side, in particular the underside, in the direction of the bottoms (123) of the cavities (120) to such an extent that, when the textile fabric (160) is arranged on the second side, at least some of the cavities (120) are fluidically connected to the textile fabric (160).

15. Microfluidic device (200), in particular according to claim 8 or 9, configured to carry out a method (500) according to one of claims 9 to 12.

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