Well-bearing substrate with recesses for the accommodation and microfluidic analysis, in particular counting, of biological cells, in particular circulating tumor cells

Abstract

The invention relates to a substrate (100) for the accommodation and microfluidic analysis, in particular counting, of biological cells (40), in particular circulating tumor cells (40), the substrate (100) having on an upper face (110) wells (120) for accommodating the cells (40), walls (121, 122) and / or bottoms (123) of at least some wells (120) having recesses (130) (130), in particular pores, for assisting removal of accommodated cells (40) from the wells (120). The invention further relates to a microfluidic device (200), and to a method (500) for the accommodation and microfluidic analysis of biological cells (40) by means of such a substrate (100) and to a production method (600) for such a substrate (100).

Description

[0001] Description

[0002] title

[0003] Cavity substrate with recesses for the absorption and microfluidic analysis, in particular counting, of biological cells, especially circulating tumor cells

[0004] State of the art

[0005] 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.

[0006] 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.

[0007] 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.

[0008] Disclosure of the invention

[0009] Advantages of the invention

[0010] Against 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 bottoms of at least some cavities have recesses, in particular pores, to support removal of received cells from the cavities.

[0011] 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 can be used and referred to as a sedimentation array as described above and can be 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.

[0012] Alternatively, the substrate can comprise, for example, germanium, aluminum, titanium, tungsten or be made 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 therefore 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, in particular in the case of silicon, the recesses are formed in the cavities, in particular by porosifying at least part of the substrate comprising the affected cavities.Porosification of the substrate is understood in particular to mean an etching process, in particular an electrochemical etching process in electrolytes containing hydrofluoric acid or alternatively a currentless 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 porosified in this way can be largely freely selected over the process time and is well-defined in a time-controlled manner. As an alternative to the porosification of silicon, the substrate to be porosified can, as described above, comprise, for example, germanium, aluminum, titanium, or tungsten, or consist of one of these materials.

[0013] Due to the recesses in the walls and / or floors of the cavities, adhesion, in particular sticking, is advantageously achieved on the surface due to the associated structures, in particular nanostructures, which has the advantage that the absorbed cells can be more easily removed from the cavities if necessary. Furthermore, it is advantageous that, due to the lack of adhesion, the absorbed cells can be analyzed from all sides 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 analyzing, in particular counting, biological cells, in particular circulating tumor cells, using such a substrate, wherein at least some of the cells absorbed into the cavities, preferably via sedimentation, are removed from the cavities again after a preferential analysis, in particular counting.

[0014] 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 the counting of the collected cells has been completed, for example in order to subject the cells to further analysis outside the cavities depending on the counting results.

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

[0016] 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 in particular be pores. At least one part, in particular a layer, of the substrate comprising at least some of the cavities can be porous for this purpose, in particular meso- and / or nanoporous. This part of the substrate thus preferably has a sponge-like structure, which can be traversed by channels created by the porosification, the ends of which open into the cavities preferably forming recesses according to the invention.In the case of porous silicon and similarly acting materials, the high biocompatibility is advantageously exploited, whereby the porosity significantly inhibits the growth or adhesion of biological substances, especially cells or proteins, and thus prevents "clogging" or "biofouling." 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, thus they do not adhere, but remain mobile in the recesses. They can therefore be expelled from the cavities again if necessary, even by the application of only small forces.

[0017] According to a particularly advantageous development of the invention, at least one recess, preferably at least one recess in each case in a plurality of cavities, extends as a through-channel to a second side of the substrate, in particular to an underside of the substrate opposite the top side. The upper end of the through-channel preferably ends in the bottom of the respective cavity, so that the bottom 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 accommodated in the cavities being entrained due to the smaller diameter of the through-channels compared to the diameter of the cells. Furthermore, the through-channels can be used to guide a displacement medium into the cavities to remove the accommodated 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 chlorinated hydrocarbon or an aliphatic hydrocarbon.

[0018] Depending on the position of the openings of the through-channels, 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, 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 diaphragm 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.

[0019] 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 porosification described above, the through-channels can preferably be pore channels, which can in particular form the sponge-like structure described above in the porosified substrate. For this purpose, the substrate can be completely porosified to produce the pore channels. Alternatively, the partially porosified substrate, in particular layer-by-layer porosified, can be ground down from a second side, in particular the underside, towards the bottoms 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 between the front and back surfaces exist via the electrolyte. For this reason, a grinding process from the back of the substrate is recommended to overcome the last non-porous portion of the substrate to the porous material, or the use of an electroless porosification process for the final portion.

[0020] In a particularly preferred development, the substrate comprises a textile fabric, wherein the textile fabric is fluidically connected to recesses of at least some cavities, in particular via the corresponding through-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 border the underside of the substrate or be arranged on the underside. A textile fabric can be understood, in particular, as an arrangement of several threads and / or fibers, both also referred to as fibers below, in particular comprising naturally occurring biogenic or mineral fibers (natural fibers) and / or created organic or inorganic fibers (synthetic fibers). The arrangement can have a systematic, in particular a regular arrangement, at least in part or entirely.However, the fibers can also be arranged at least partially without any predetermined regularity and, in particular, can be entangled or woven with one another. Alternatively or additionally, the textile fabric can comprise 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 one another 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 through the recesses, but preferably allows displacement medium, for which the textile fabric is permeable, to be transported through the textile fabric via the recesses, in particular via the through-channels, into the cavities. In the case of the at least partial porosification of the substrate (described above), the textile fabric preferably borders at least partially on a porous part of the substrate. According to a particular embodiment, the textile fabric forms the floor of at least some of the cavities. In other words, in this embodiment, instead of a floor made of substrate material, a floor made of the textile fabric is provided, while the walls are preferably still formed by preferably porosified or non-porosified substrate material.The textile fabric can therefore completely replace the porous substrate, in particular silicon, on the bottom of the cavities. In general, in a further aspect, the invention also relates to a substrate for receiving and microfluidically analyzing, in particular counting, biological cells, in particular circulating tumor cells, wherein the substrate has cavities on a top 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 recesses described above, in particular in the form of pores and / or through-channels, are preferably developments of this second aspect.

[0021] According to a particularly advantageous development of the invention, 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 together with the fluid absorbed in the textile fabric enables 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 fabric such as GoreTex®, a gas, a gas mixture or preferably a fluorocarbon can be used as the heat transfer fluid. If the textile fabric comprises non-fluorinated fibers, the heat transfer fluid can in particular comprise aliphatic hydrocarbons. This heat transfer fluid can preferably also be used as a displacement medium for the removal of the cells, as described above.For this purpose, more of 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, thereby removing the contents of the cavities, in particular the cells. Alternatively or additionally, a second displacement medium can be introduced into the textile fabric, in particular pumped in, to force the fluid into the cavities as the first displacement medium. Brief description of the drawings.

[0022] Embodiments of the invention are shown schematically in the drawings and explained in more detail in the following description

[0023] It shows

[0024] Figure 1a shows a flow diagram of an embodiment of the method according to the invention for the recording and microfluidic analysis of biological cells,

[0025] Figure 1 b is a flow diagram of an embodiment of the inventive manufacturing method of a substrate according to the invention,

[0026] Figure 2 shows an embodiment of the device according to the invention and

[0027] Figures 3-8 show embodiments of the substrate according to the invention, preferably as part of the device according to the invention.

[0028] 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), schematically illustrated in vertical cross-section in Figure 3a. 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 formed 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. Figure 4 shows an enlarged view of a portion of the substrate 100, schematically illustrating that the bottom 121 and walls 122, 123 of the cavities 120 have recesses 130, which serve to reduce adhesion or sticking of the captured cells 40 in the cavities 120. Preferably, the recesses 130 are smaller than the target cells 40 to be captured in the cavities 120, as shown.

[0034] These recesses 130 can, during the second step 602 of the manufacturing method 600 according to the invention, be formed, for example, as pores. A layer 140 of the substrate can be rendered porous, with this layer at least partially and preferably (as shown in Figure 4) completely encompassing the cavities 120. If the substrate comprises silicon (Si for short) or is made of silicon, the surface and / or a near-surface zone of the substrate 100 can, after the cavities 120 have been produced, preferably completely and over the entire surface, be electrochemically converted into porous silicon. In this process, a certain Si layer thickness is converted by electrochemical anodization into a nanoporous or mesoporous Si layer, which is traversed by a plurality of nanoscale channels 130 and forms a sponge-like silicon structure.In Figure 4 and also Figures 5 to 7, the pores 130 are only partially drawn and are intended to illustrate that, depending on the etching process, the pores here extend mainly vertically from the top side 110 to the bottom 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 the addition of alcohols such as methanol, ethanol, or isopropanol to reduce surface tension, is well understood and can be carried out 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 passing electrical current through the silicon wafers 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 future surface 110 of the substrate 100) with the cavities 120 must be anodically polarized 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 located on the underside of the wafer by means of p-doping of the underside of the wafer 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 of more than 100 million per year.

[0035] 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.

[0036] The cells 40 collected in the cavities, which come into contact with the porous base 121 on which they come to rest and possibly also with the porous side walls 122, 123, do not form substantial bonds to the 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 action 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, gravity, which caused the cells 40 to sink into the cavities 120 during the sedimentation phase 503, can be used again, for example.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 performed cell count 504 (for example a CTC count) in order to supply the cells 40 for further analysis outside the device 200.

[0037] 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 several hundred μm 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 shown schematically in Figure 5, in particular nanoscale 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] Alternatively or in addition to the change in position described above, in the fifth step 505, a displacement medium, in particular a fluid, can also be used to remove 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 also a chlorinated hydrocarbon 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 rinse 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 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.

[0039] In both embodiments according to Figures 4 and 5, the substrate 100, in particular the silicon substrate, can be ground ("re-ground") from the underside 150 toward the top side 110, before and / or after the porosification to create the recesses 130. In Figures 4 and 5, the dashed line 300 shows by way of example that this re-grinding preferably takes place to such an extent that the layer 140 with completely porosified substrate material or a porous substrate layer of reduced thickness remains below the cavities 120. I

[0040] Figures 6 and 7 show exemplary embodiments of an advantageous further development of the invention, in which 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, for example, comprise 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 6, 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 adjoin the cavities 120 and thus form the bottom 121 of the cavities 120, as shown in Figure 7. For example, the textile fabric can have a thickness of 100-500 μm. These embodiments also offer the particular advantage of making it easy to fill the cavities 120 with an aqueous sample medium, as gas bubbles in the cavities 120 can easily pass through the porous substrate residue layer toward the underside 150 and the textile fabric 160 until the aqueous sample fluid reaches them and can penetrate practically not at all (with GoreTex®) or only with difficulty (with non-fluorinated synthetic fiber fabric). Cells 40 that are deposited in the cavities 120 generally do not adhere to perfluorinated fabric (GoreTex®).When using non-fluorinated synthetic fiber fabric, the degree of cell adhesion suppression 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 preferred case shown in Figure 6, cell adhesion 40 is virtually eliminated by the porous residual substrate layer.

[0041] 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 6) 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.

[0042] 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 toward the cavities 120. When using GoreTex® or other perfluorinated textile fabrics, a gas, a gas mixture, or a fluorocarbon is preferred, whereas when using non-fluorinated synthetic fiber fabrics, a low-viscosity oil or aliphatic hydrocarbon with low surface tension is advantageous. Preferably, the above-described fluid for heat transfer 161 already present in the textile fabric 160 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 forcing the fluid 161 present therein into the cavities 120.

[0043] The use of a porous substrate, particularly silicon, can therefore be used either as an alternative to or in addition to a synthetic fiber fabric finish on the underside of the substrate. The advantages of both solutions can thus be combined in a variety of ways. According to a further exemplary embodiment, it is also possible to dispense with the porosification of the substrate 100 entirely and to use only the synthetic fiber fabric finish for the cavities 120, as shown in Figure 8, 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 wells 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.

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 an upper side (110) for receiving the cells (40), characterized in that walls (121, 122) and / or bottoms (123) of at least some cavities (120) have recesses (130), in particular pores, to support removal of received cells (40) from the cavities (120).

2. 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) 3. 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 porous, in particular meso- and / or nanoporous, and wherein at least some of the recesses (130) are formed as pores.

4. Substrate (100) according to one of the preceding claims, wherein at least one recess (130), preferably at least one recess (130) in each case in a plurality of cavities (120), extends as a through-channel (124) to a second side (150) of the substrate (100), in particular a bottom side (150) of the substrate (100) opposite the top side (121).

5. Substrate (100) according to one of the preceding claims, wherein the substrate (100), in particular on the underside (150) of the substrate (100), has 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 at least some of the recesses (130).

6. Substrate (100) according to claim 5, wherein the textile fabric (160) forms a bottom (121) of at least some of the cavities (120).

7. Substrate (100) according to claim 5 or 6, wherein the textile fabric (160) comprises a fluid (161) for heat transfer.

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 at least some of the recesses (130), 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. Method (500) according to claim 10 or 11, wherein the removal of the cells (40) is carried out by introducing a displacement medium, in particular a gas or a Liquid, in particular a fluid (161) located in the textile fabric (160) for heat transfer, is supported or carried out 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 preferably is moved through the bottom (121) of the cavities (120) into the cavities (120).

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). • Forming (602) the recesses (130) in the cavities (120), in particular by porosifying at least part of the substrate (100) 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) until at least some of the recesses (130) (130) as through-channels (124) fluidically connect at least some cavities (120) to the underside.

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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