Electrophysiological measuring device and measuring method for recording at least one electrical measurement value on a biological cell sample

Description

[0001] The invention relates to electrophysiological measuring devices and electrophysiological measuring methods for detecting at least one electrical measurement value from a biological cell sample, in particular to a measuring device and a measuring method for detecting an action potential at at least one cell membrane of the cell sample and / or an ion current through at least one cell membrane of the cell sample. Substrates for receiving biological cells and their applications are also described. The invention is applicable to the handling and, in particular, characterization of biological cell samples, in particular individual cells or cell aggregates, in pharmacology, medicine, biochemistry, and / or biomedicine.

[0002] In this description, reference is made to the following prior art, which represents the technical background of the invention: [1] EP 1 040 349 B1; [2] OP Hamill et al. in "Pfluger's Arch." 1981 Aug; 391(2):85-100; [3] EP 1 349 916 A2; [4] J. Abbott et al. in "Acc. Chem. Res." 51 (2019) 600-608; [5] A. Cerea et al. in "Lab Chip" 18 (2018) 3492-3500; [6] X. Xie et al. in "ACS Nano" 9 (2015) 11667-11677; [7] X. Xie et al. in "ACS Nano" 7 (2013) 4351-4358; [8] K. Li et al. in "Comprehensive Biotechnology" Chapter 5.11, pp. 125-139 (Elsevier BV 2011, DOI: 10.1016 / B978-0-08-088504-9.00497-9); [9] MR Angle et al. in "Biophysical Journal" 107 (2014) 2091-2100;

[10] "Nanostructured metal electrodes for functional coupling to neuronal cells", Dorothea Brüggemann, dissertation, RWTH Aachen, Schriften des Forschungszentrums Jülich, Volume 9, ISBN 978-3-89336-627-9;

[11] WO 2010 / 031506 A1;

[12] AM Shum et al. in "Adv. Mater." 29 (2017) 1602448;

[13] EP 1 953 812 A1;

[14] C. Morez et al. in "Biomaterials" 70, 94-104;

[15] MA Bucaro et al. in "ACSNANO" 6 (2012), 6222-6230;

[16] DE 10 2017 130 518;

[17] R.Wang et al. in "Scientific reports" 8 (2018) Article number: 14205;

[18] "Development of an autocontractile heart muscle model for functional drug and toxin research" Matthias Goßmann, Dissertation, University of Duisburg-Essen, 2015, (https: / / duepublico2.uni-due.de / servlets / MCRFileNodeServlet / duepublico_ derivate_00040212 / DissGossmann.pdf;

[19] M. Goßmann et al. in "Cell. Physiol. Biochem." 2016; 38:1182 - 1198;

[20] E. Musk et al. "An integrated brain-machine interface platform with thousands of channels" (http: / / dx.doi.org / 10.1101 / 703801);

[21] DE 198 27 957 A1;

[22] US 2006 / 0 163 062 A1;

[23] YI Zilberter et al. in "Pflugers Arch." 1982 Aug, 394(2):150-5; and

[24] US 2006 / 129043 A1. .

[0003] Electrophysiological studies on cell samples from at least one biological cell or cell aggregates include, in particular, the measurement of ion currents through the cell membrane ("patch-clamp" measurement) or the measurement of action potentials at the cell membrane ("current-clamp" measurement). In the older "voltage-clamp" method, a compensating current is introduced into the sample to keep the membrane voltage constant, whereas in "patch-clamp" measurements, the injection of a compensating current during the measurement can be omitted due to the low current amplitudes. It is known to perform electrophysiological measurements at pipette tips or with planar measuring devices in which a single cell or a reconstituted membrane system is positioned on a pore in a flat surface ("patch pore") ([1], [2]).Multiple pores can be present on a surface, allowing multiple single cells to be examined simultaneously using the patch-clamp technique ([2]) (parallelization of the examination). The (multiarray) patch-clamp method or patch-clamp chips are described, for example, in [1] and [3]. In patch-clamp measurements, the cell membrane of a single cell or the membranes of several cells form a firm connection with the edge of a suction pore (also called a patch pore), which is connected to a suction device. The so-called seal resistance is formed at the edge of the suction pore. In a variant of patch-clamp measurements, the so-called "cell-attached" measurement, the membrane in the pore is not destroyed but remains intact (see

[23] ). A disadvantage of these measurements is that the membrane potential (unlike the so-called "whole cell" measurement) cannot be recorded.

[0004] The use of nanoelectrode arrays has been proposed to further parallelize electrophysiological studies (see, for example, [4] to [8]). A nanoelectrode array comprises a large number of compact ([4], [8]) or hollow ([5] to [8]) electrode tips with typical diameters in the sub-micrometer range, which protrude from a solid substrate, for example as a matrix arrangement. In order to perform an intracellular measurement (measurement inside the cell), i.e., to establish direct electrical contact between the electrode and the cytosol inside the cell, the electrode tips must penetrate the cell membrane. Otherwise, without the electrode tips penetrating the cell interior, the cytosol would be isolated from the electrode tip by the cell membrane, so that in practice only an extracellular measurement (measurement on the outside of the cell) could be performed.However, the extracellular measurement is less meaningful and less accurate than the intracellular measurement due to the relatively low potentials.

[0005] Penetration of the cell membrane can occur spontaneously or be induced by electroporation [4]. Spontaneous penetration has the disadvantages that it is a rare, non-reproducible event, and the electrode often penetrates the cell only insufficiently. Electroporation, on the other hand, can be associated with undesirable influences on the cell and thus on the measurement results of the electrophysiological study. Hollow nanoelectrodes of the nanoelectrode arrays from [5] to [8] allow not only electrophysiological measurements or sensory tasks but also the delivery of biomolecules into the cell interior. In addition to the aforementioned disadvantages regarding the penetration of the electrodes into the cells, the hollow nanoelectrodes have further disadvantages due to the limited miniaturization potential and complexity of the substrate structure.

[0006] The measurement of the force required to penetrate the cell membrane with a nanoelectrode using atomic force microscopy (AFM) techniques is described in [9]. The electrode under investigation is mounted on a flexible AFM carrier and inserted into a cell. While this technique provides information about the forces required for penetration, it would be unsuitable for practical, routine electrophysiological studies because it is limited to the application of AFM technology and measurements with single electrodes.

[0007] Further applications of nanoelectrode arrays are described in

[10] and

[11] . According to

[10] , extracellular signals from biological cells are recorded using nanostructured metal electrodes. These nanostructured metal electrodes are unsuitable for intracellular measurements. In

[11] , it is proposed to exploit electrophysiological potentials to generate electrical currents. To promote the penetration of the nanoelectrodes into the cells, the tips of the nanoelectrodes are provided with a coating that activates endocytosis of the membrane and thus facilitates the uptake of the electrodes into the interior of the cells. This technique is unsuitable for electrophysiological studies because the endocytosis coating can have undesirable effects on the cells.

[0008] Another disadvantage of conventional nanoelectrode arrays can arise from natural cell movement, which adherent biological cells can perform due to ongoing rearrangement of their cell-substrate contacts. Cells can migrate across a substrate with nanoelectrodes during measurement, distorting the measurement result. This natural cell movement can be restricted by an adhesion-reducing substrate coating. However, the substrate coating has the disadvantage that it can influence the physiology of the cells.

[0009] The interest in parallelizing cell investigations is not only to increase the number of simultaneous measurements. Rather, by arranging a large number of biological cells on a substrate with a structured surface, physiological conditions, such as those found in tissue, can be simulated. Structured substrates for accommodating a large number of biological cells, as described, for example, in

[12] , allow the observation of biochemically induced cell changes and their transmission from cell to cell. In this case, too, cell movements should be prevented as much as possible, which is only inadequately possible with the substrates known from

[12] . Deformable substrates are described in

[13] that can accommodate biological cells, but would only be suitable for mechanical displacement of biological cell samples.From

[14] and

[15] , structured substrates with so-called microgrooves are known. These are intended to influence the cultivation of adherent cells, but in practice, they may have limited effectiveness. In general, it can also be disadvantageous if conventional substrates carry coatings, such as Matrigel or fibronectin, which can induce undesirable biochemical changes in cells.

[0010] Further electrophysiological studies on biological cells are described in

[21] and

[22] , where the cells are sucked and held using negative pressure on electrodes with typical diameters in the mm or µm range. In

[21] , the use of a microelectrode with a diameter above 10 µm is proposed, which is located centrally inside a cylindrical electrode casing in which a negative pressure is generated and which forms a suction device. According to

[22] , the electrode is arranged centrally in a suction opening and protrudes in front of it. Due to the relatively large electrode diameters, these techniques can have disadvantages both in terms of the space required by the electrodes and in terms of electrophysiological signal recording. The technique according to

[21] has limited applications, in particular because cell-attached measurements are not possible.

[0011] An electrophysiological measuring device is known from

[24] in which a cell is held under the action of suction pressure on a pore containing a spike-shaped electrode. Measures for stable support of the cell on the pore are not disclosed in

[24] .

[0012] The object of the invention is to provide an improved electrophysiological measuring device and / or an improved electrophysiological measuring method for recording at least one electrical measurement value from a biological cell sample, with which disadvantages and limitations of conventional techniques can be avoided. In particular, the invention is intended to enable electrophysiological investigations with increased accuracy, reproducibility, and reliability, be suitable for intracellular measurement on cells, permit routine applications, and / or have an expanded range of applications. According to a further aspect, the invention is intended to provide an improved electrophysiological measuring device and / or an improved electrophysiological measuring method, enabling better adhesion of cells to substrate surfaces.

[0013] These objects are achieved by devices and methods having the features of the independent claims. Advantageous embodiments and applications of the invention are set forth in the dependent claims.

[0014] According to a first general aspect of the invention, the above object is achieved by an electrophysiological measuring device configured to record at least one electrical measurement value from a biological cell sample. The electrophysiological measuring device comprises a substrate device configured to receive the cell sample. The cell sample comprises a single biological cell or a plurality of cells (e.g., a cell cluster, cell aggregate, or tissue section, in particular a cell aggregate with cells connected by nexus contacts). The substrate device has a substrate with a substrate surface for the adherent positioning of cells. The substrate surface preferably has a planar shape, thereby defining a reference plane (hereinafter also: xy plane) of the substrate, wherein the substrate surface itself can be planar or structured with elevations and / or depressions.

[0015] Furthermore, the substrate device is configured to provide at least one liquid film, preferably a liquid layer or a liquid bath, for covering the cells and is preferably, but not necessarily, formed as part of a chamber (vessel) for this purpose. The chamber can be closed (e.g., a dish) or configured for flow (e.g., a channel). The chamber can have a projection in a chamber wall above the substrate device for securing the cell sample. The substrate device can be one-part or multi-part and / or at least partially formed by parts of the measuring device that are configured for further functions besides receiving the cell sample. The substrate device comprises, in particular, a substrate that, in various configurations, comprises a single substrate body for receiving the cell sample or can be combined with other components, such as membranes.The substrate has a substrate surface, which is preferably made of a cell-compatible material and, for example, bears a cell-compatible coating. The substrate surface, in particular the coating, advantageously fulfills a dual function with respect to insulating the conductive tip of at least one nanoelectrode and ensuring direct, tight contact with the cell membrane.

[0016] The electrophysiological measuring device further comprises an electrode device with at least one, preferably a plurality of nanoelectrodes (in particular at least two nanoelectrodes). The electrode device has a single or a plurality of nanoelectrodes. If features are described below with reference to multiple nanoelectrodes, these features preferably also apply in the same way to variants with a single nanoelectrode. Each nanoelectrode extends straight in a longitudinal direction (hereinafter also: z-direction) from an electrode carrier to a free electrode tip (free end). The diameter of the nanoelectrodes, in particular at their free electrode tip, which protrudes into the cell sample, is less than 1 µm, preferably less than or equal to 800 nm, e.g., less than or equal to 500 nm, and / or at least 50 nm.The longitudinal length of the nanoelectrodes is preferably at least 100 nm, more preferably at least 1 µm, and / or at most 10 µm, more preferably at most 3 µm.

[0017] The electrode device and the substrate device are arranged such that the nanoelectrodes can be inserted intracellularly into the cell sample once the cell sample has been received by the substrate device. The nanoelectrodes are configured such that they can protrude into at least one of the cells of the cell sample. The electrode device and the substrate device are connected to one another. Preferably, the nanoelectrodes are arranged such that the longitudinal direction of the nanoelectrodes runs perpendicular to the reference plane of the substrate surface. In at least one operating state of the measuring device, the nanoelectrodes are positioned relative to the substrate surface such that the free electrode tips are at a perpendicular distance from the substrate surface, so that the nanoelectrodes are in direct contact with the cytosol inside the cell sample on the substrate surface.The vertical distance from the substrate surface is preferably at least 1 µm and / or at most 1 mm, but can also be selected in the range of less than 1 µm. The nanoelectrodes can be mounted on a shaft and form columns or taper to a point. The shaft can be formed by a cylinder or truncated cone with a height of 1 µm to 50 µm, onto which the nanoelectrodes are placed. However, the shaft can also be taller.

[0018] Preferably, the electrode device of the electrophysiological measuring device can be coupled to a measuring device or is permanently connected. The measuring device is configured to detect electrical quantities, in particular voltages and / or currents, at the nanoelectrodes and to determine electrophysiological measured values, such as resting potentials, action potentials, or ion currents through cell membranes. Preferably, the measurement of ion currents through the cell membrane ("patch-clamp" measurement) or the measurement of action potentials at the cell membrane ("current-clamp" measurement) is carried out. Simultaneous or sequential measurements of both action potentials and ion currents are also possible using the cell-attached patch clamp method on a cell sample.

[0019] According to the invention, the electrophysiological measuring device is equipped with an actuating device designed for mutual movement of the nanoelectrodes and / or the cell sample along the longitudinal direction of the nanoelectrodes. The mutual movement is configured such that the nanoelectrodes can penetrate into the cell sample when the cell sample is received by the substrate device. The actuating device acts on the nanoelectrodes, in particular their electrode carriers and / or the nanoelectrodes themselves, and / or the substrate device, in particular the substrate or a lateral chamber wall, so that the nanoelectrodes are actively moved toward the cell sample and / or the cell sample toward the nanoelectrodes. The actuating device acts such that the nanoelectrodes penetrate the cell sample simultaneously. The relative movement preferably extends over a distance in the range of 100 nm to 5 mm.

[0020] Advantageously, the provision of the actuator of the electrophysiological measuring device overcomes numerous limitations of conventional techniques. The nanoelectrodes, particularly their electrode tips, are precisely positioned within the biological cell sample. The introduction of the nanoelectrodes into the cytosol is triggered by actuating the actuator. The measurement is not dependent on spontaneous processes or processes triggered by electrode coatings. The accuracy, reproducibility, and reliability of the measurement are improved by positioning the nanoelectrodes within the cell sample. The inventors have discovered that biological cell samples allow the simultaneous introduction of a large number of nanoelectrodes with penetration of the cell membrane without damaging the nanoelectrodes.By allowing the actuator to move the nanoelectrodes and / or the cell sample relative to each other along the longitudinal direction of the nanoelectrodes, unwanted lateral forces that could bend the nanoelectrodes are avoided or reduced to a negligible level. The inventors have further discovered that the cell membrane has a stabilizing effect on the nanoelectrodes, allowing them to be guided from the cell membrane into the cytosol.

[0021] According to a second general aspect of the invention, the above object is achieved by an electrophysiological measuring method in which at least one electrical measurement value is recorded intracellularly on a biological cell sample. The measuring method, which is preferably carried out with the electrophysiological measuring device according to the first general aspect of the invention, comprises the following steps. First, the cell sample, comprising a single cell or a plurality of cells, is arranged on a substrate device. The arrangement of the cell sample on the substrate device comprises, for example, positioning a cell suspension on a substrate of the substrate device, optionally followed by culturing the cell sample. Alternatively, the arrangement of the cell sample comprises positioning the substrate device in a cell suspension, followed by transferring at least one cell to the substrate device.Subsequently, or during the transfer of the at least one cell to the substrate device, a plurality of nanoelectrodes of an electrode device are positioned such that the nanoelectrodes, in particular their electrode tips, protrude intracellularly into the cell sample. The nanoelectrodes extend parallel to one another in a longitudinal direction and perpendicular to a reference plane representing a lateral extent of the substrate surface. According to the invention, the positioning of the nanoelectrodes comprises moving the nanoelectrodes and / or the cell sample along the longitudinal direction of the nanoelectrodes using an adjusting device until the nanoelectrodes protrude into the cell sample and form direct electrical contact with the cell interior. The at least one electrical measurement value is then measured on the cell sample.

[0022] Penetration of cells in the cell sample can occur upon actuation of the actuator in such a way that the nanoelectrodes first contact and deform the cell membrane with their tips (destabilization) and then penetrate the cells through a pulsed forward movement. The pulsed forward movement can be achieved by suitable control of the actuator, in particular by controlling the actuator via the drive unit.

[0023] The provision of the adjustment device offers the advantage that the cell sample can be spatially separated from the nanoelectrodes prior to measurement. This prevents unintentional penetration of the nanoelectrodes into the cells or reduces it to a negligible level. Parts of the measuring device, in particular the substrate feeder and / or the adjustment device, form a separation device designed to spatially separate the cell sample from the nanoelectrodes prior to operation of the adjustment device. Actuation of the adjustment device establishes a temporal reference point for the electrophysiological measurement, so that it can, in particular, include the recording of a time-dependence of electrophysiological variables.

[0024] Advantageously, a number of variants are possible for implementing the adjusting device in the electrophysiological measuring device according to the invention, as shown below.

[0025] According to the invention, the actuating device comprises a suction device as an actuator and drive unit, which is configured to draw the cell sample toward the nanoelectrodes. The suction device is formed by at least one suction opening and a pumping device (suction pump) that is connected to the space above the substrate surface via the at least one suction opening in the substrate device. The substrate is equipped with the at least one nanoelectrode. In the first embodiment, the nanoelectrodes are preferably fixed to the substrate, arranged protruding from the substrate, or recessed into the substrate. When the pumping device applies a negative pressure relative to the ambient atmospheric pressure to the at least one suction opening, the cell sample and / or a surrounding suspension liquid is drawn toward the substrate.The at least one suction opening is formed such that when the at least one suction opening is subjected to negative pressure, the cell sample is moved towards the substrate surface and with at least one directional component parallel to the longitudinal direction of the nanoelectrodes. When a nanoelectrode comes into contact with a cell, the nanoelectrode is guided into the cell by the suction movement. The first embodiment of the invention has the particular advantages that the measuring device has a simple structure and the actuating device is easy to control. The power of the pump device is preferably variable and controllable so that a predetermined suction process can be carried out which enables reliable penetration of the membrane of the cell sample. The suction process takes place, for example, in such a way that in a first phase with a lower suction pressure a slower movement of the cell sample is effected than in a second phase with a higher suction pressure.The suction device can be used to move cell samples of various sizes. The distance traveled by the cell sample can be adjusted by the duration of the pump's operation. The suction device can also capture non-adherent cells in the space above the substrate.

[0026] According to the invention, a chamber having a chamber wall with a conical shape narrowing towards the substrate is provided above the substrate device, wherein the chamber is designed to receive a culture medium and the cell sample in the form of a cell aggregate, and above the substrate device, a projection for fixing the cell sample is provided in the chamber wall at the lower end of the chamber.

[0027] In the invention, the nanoelectrodes are preferably arranged protrudingly on the substrate of the substrate device, wherein the at least one suction opening is preferably arranged directly adjacent to the nanoelectrodes. In other words, the nanoelectrodes are arranged outside the at least one suction opening at its edge (edge-side nanoelectrodes). Preferably, the nanoelectrodes and the at least one suction opening are located in one plane. The cell membrane of a cell sample is not penetrated in the center of the suction opening, but laterally next to the suction opening. Advantageously, the arrangement at the edge of the at least one suction opening simplifies the movement of the cell sample parallel to the longitudinal direction of the nanoelectrodes. Furthermore, the use of edge-side nanoelectrodes simplifies the manufacture of the measuring device according to the invention and in particular the contacting of the nanoelectrodes.

[0028] Advantageously, the at least one suction opening can be equipped with an adhesive surface at its edge, where the nanoelectrode(s) are located, which promotes cell adhesion. A sealing resistor (particularly in the high mega- or giga-ohm range) is formed with the cell sample at the adhesive surface of the peripheral edge. This improves the simultaneous or sequential measurements of action potentials and ion currents on a cell sample using the cell-attached patch clamp method. The use of edge-attached nanoelectrodes eliminates limitations of conventional cell-attached measurements, and the membrane potential can be directly determined and used for the simultaneous patch clamp measurement on the cell sample.

[0029] Particularly preferably, the at least one suction opening is provided in the substrate between the nanoelectrodes, so that when the cell sample is aspirated, the adjacent nanoelectrodes penetrate the cell sample. The nanoelectrodes can, for example, be arranged along a circle around a central suction opening. For example, a plurality of suction openings is provided in the substrate between the positions of the nanoelectrodes. A further advantage of the suction device is that components are available from fluidic microsystem technology with which the suction device of the electrophysiological measuring device can be implemented.

[0030] According to an advantageous variant, the at least one nanoelectrode can be arranged at an angle toward the at least one suction opening. This angle can be advantageous for the reliable and deformation-free introduction of the nanoelectrode(s) into the cell sample.

[0031] Preferably, each nanoelectrode is connected to a measuring device via a connecting line, wherein the connecting line is arranged outside the at least one suction opening, at the edge of which the nanoelectrode is positioned. This advantageously simplifies the design of the measuring device.

[0032] The shape of the suction opening can advantageously be selected depending on the specific application of the invention. The suction opening can, for example, be circular or angular. Alternatively, the suction opening can have an elongated slit shape. According to a further advantageous variant of the invention, the suction opening alternatively or additionally comprises an annular opening arranged in the substrate device and connected to the pump device. The annular opening is arranged such that it surrounds at least one group of nanoelectrodes along the reference plane of the substrate. Advantageously, the annular opening offers the possibility of forming a unified flow towards the nanoelectrodes in the space above the substrate.

[0033] According to a further modification of the invention, the nanoelectrodes can be arranged in at least one of the suction opening(s) in the substrate. At least one nanoelectrode can be provided in each suction opening. In this case, for example, a single nanoelectrode or multiple nanoelectrodes extend in each suction opening with the longitudinal direction perpendicular to the reference plane of the substrate. When a negative pressure is applied to the suction opening, the cell sample is drawn to the suction opening, with the electrode tip penetrating into the interior of the cell sample. The cell sample can be partially drawn into the suction opening, where the nanoelectrodes penetrate the cell sample. The positioning of the nanoelectrodes in at least one of the suction openings has the particular advantage that the nanoelectrodes in the suction openings receive mechanical protection.Furthermore, the movement of the cell sample parallel to the longitudinal direction of the nanoelectrodes is facilitated.

[0034] Preferably, the at least one nanoelectrode extends parallel to an inner wall of the suction opening in the substrate device. The inner wall preferably extends perpendicular to the reference plane of the substrate. This advantageously stabilizes the nanoelectrodes during cell penetration.

[0035] Electrode tips of the nanoelectrodes can protrude above the substrate surface or be recessed into the suction opening. In the latter case, the at least one nanoelectrode is arranged such that its free end is at a predetermined perpendicular distance from the surface of the substrate device.

[0036] According to a further advantageous variant, the at least one suction opening can be free of reference electrodes for the intracellular detection of the at least one electrical measurement value. Advantageously, no reference electrode is required for the cell-attached measurement provided according to the invention, since the intracellular potential of the cell sample can be used as the reference potential for the measurement.

[0037] In the variants of the invention described above, the suction device can act directly on the cell sample. According to a further modification of the first embodiment, the action can be provided on a deformable carrier membrane designed to receive the cell sample. The deformable carrier membrane is part of the substrate device. The carrier membrane, which is, for example, a silicone membrane (e.g., ultra-thin and hyper-elastic silicone membrane, see, for example,

[18] ) or made of collagen, has such a small thickness that it can be penetrated by the nanoelectrodes when the suction device is actuated. The thickness of the carrier membrane is selected, for example, in the range of 1 µm to 30 µm.The deformable support membrane is positioned at a distance above the substrate with the nanoelectrodes such that when negative pressure is applied to at least one suction opening, the deformable support membrane with the cell sample is moved toward the substrate. The cell sample and the support membrane move parallel to the longitudinal direction of the nanoelectrodes. The nanoelectrodes penetrate the support membrane and the cell membrane of the cell sample.

[0038] According to a further modification, the substrate device comprises a mouth region of a suction tube device, such as a pipette tip (not an embodiment of the invention). The mouth region is a free end of the suction tube device, at which it has a mouth opening. The nanoelectrodes are arranged protrudingly at a peripheral edge of the mouth opening. The nanoelectrodes can carry insulating layers that leave the electrode tips of the nanoelectrodes exposed, or they can carry a viscous coating, or they can be uncoated. The longitudinal direction of the nanoelectrodes runs parallel to the axial direction of the suction tube device. In this variant, the suction opening is formed by the mouth opening of the suction tube device.This variant advantageously allows for combination with conventional techniques for the electrophysiological examination of cell samples, in particular with the conventional patch-clamp technique. Therefore, a measuring electrode configured for ion current measurement with a patch-clamp amplifier (current amplifier) ​​is particularly preferably arranged inside the suction tube device.

[0039] As an alternative to the above description, the latter variant, in which the substrate device forms the mouth region, in particular of a patch-clamp pipette, can generally be described as a novel patch-clamp pipette equipped with nanoelectrodes for cell penetration (not an embodiment of the invention). Accordingly, a patch-clamp pipette with nanoelectrodes at the pipette tip is described, wherein the patch-clamp pipette is configured for connection to a measuring device.

[0040] In addition, the actuating device can comprise an electrode drive device as an actuator and drive unit, which acts on the electrode device and is configured for introducing the nanoelectrodes into the cell sample (not an embodiment of the invention). The substrate, in particular the substrate body, has a plurality of electrode openings, in each of which one of the nanoelectrodes is arranged. In this variant, the drive device is configured for mechanically advancing the nanoelectrodes through the electrode openings until the electrode tips protrude above the substrate surface into the cell sample. Alternatively, the electrode device can be connected to the substrate device such that the nanoelectrodes are arranged in the space above the substrate surface, wherein the electrode tips are arranged at a distance from the substrate surface and point towards the substrate surface.In this variant, the electrode drive unit is configured for mechanically advancing the nanoelectrodes toward the substrate surface until the electrode tips protrude into the cell sample on the substrate surface. This variant has the advantage that the nanoelectrodes can be adjusted using the adjustment unit and inserted into the cell sample, which is fixed (adherently) arranged on the substrate unit. The drive unit allows the penetration depth of the nanoelectrodes into the cell sample to be adjusted with increased precision and reproducibility.

[0041] According to a particularly preferred variant, in which the nanoelectrodes are arranged on the substrate side, the substrate of the substrate device comprises a fixed, shape-retaining porous membrane. The porous membrane forms the substrate surface for receiving the cell sample. The porous membrane has openings which, like the electrode openings, are arranged in the substrate body. The drive device is arranged such that, upon actuation, it presses the electrode device towards the porous membrane, whereby the nanoelectrodes are advanced through the openings of the porous membrane into the cell sample. Advantageously, the adhesion forces that hold the cell sample to the porous membrane are greater than the propulsive forces required for the nanoelectrodes to penetrate the cell sample.

[0042] If, according to a further modification, the substrate device comprises a compressible substrate body arranged between the electrode device and the pore membrane, and the electrode drive device presses the electrode device against the substrate body, so that the substrate body is compressed between the electrode device and the pore membrane while the nanoelectrodes are advanced through the electrode openings through the substrate body and the pore membrane into the cell sample, additional advantages can be achieved with regard to the stabilization of the pore membrane during actuation of the electrode drive device (not an embodiment of the invention). The substrate body is made of a compressible plastic, such as polydimethylsiloxane (PDMS).

[0043] According to an alternative modification, the electrode openings in the substrate device can be closed with a cover membrane, wherein the electrode drive device is configured to move the nanoelectrodes through the cover membrane into the cell sample. Advantageously, the nanoelectrodes are separated from the cell sample by the cover membrane. The cover membrane forms a variant of a separation device with which the electrophysiological measuring device can be equipped (not an embodiment of the invention).

[0044] There are several possibilities for mechanically adjusting the nanoelectrodes using the electrode drive device. The electrode drive device is preferably formed by the nanoelectrodes themselves, which are made from a material that expands in response to external stimulation. In this case, the electrodes themselves form the actuator. Alternatively or additionally, the electrode drive device can be formed by the electrode carrier of the nanoelectrodes, which acts as an actuator. The electrode drive device, in particular the nanoelectrodes and / or the electrode carrier, is configured, for example, for piezoelectric, electrical, mechanical, and / or magnetic movement of the nanoelectrodes. The nanoelectrodes and / or the electrode carrier can be made from a material that is designed for expansion, in particular a change in length, of the nanoelectrodes, such as, for example,a piezoelectric material and / or an organic material, such as a polymer or resin. In these cases, an electrical control of the actuators is provided as the drive unit.

[0045] Additionally, the actuating device can comprise a substrate drive device as an actuator and drive unit, which is coupled to the substrate device and configured to pull the cell sample toward the nanoelectrodes (not an embodiment of the invention). In this case, the substrate body itself can form the actuator and be made of a material that contracts in response to external stimulation. Alternatively, the substrate body can be connected to an actuator with which the substrate body can be displaced. The nanoelectrodes are fixedly connected to the substrate device. Upon stimulation of the substrate body material or displacement of the substrate body, the cell sample on the substrate is pulled toward the nanoelectrodes, causing them to penetrate the cell sample. This variant has the advantage that the substrate body can be adjusted and moved toward the stationary nanoelectrodes using the actuating device.This allows for additional mechanical stabilization of the nanoelectrodes, e.g., with a partial embedding layer, and can simplify the connection of the nanoelectrodes to the measuring device.

[0046] Additionally, the actuating device can comprise a magnetic drive device as an actuator and drive unit, which comprises magnetic particles as an actuator, which are provided for coupling with the cell sample, and a main magnet as a drive unit, which is arranged to interact with the magnetic particles and draw the cell sample toward the nanoelectrodes (not an embodiment of the invention). Magnetic particles (magnetic beads) are advantageously available from known cell biology techniques, which are inert and can be coupled to biological cells. A further advantage of this variant is that the substrate device and the electrode device can be formed stationary without moving parts, thereby simplifying the structure of the measuring device.Finally, when measuring a cell sample with different cell types, of which only a specific group of cells of interest is equipped with the magnetic particles, it can be advantageous to selectively move only the cells of interest to the nanoelectrodes.

[0047] Additionally, the actuating device can comprise a chamber drive device as an actuator and drive unit, which is coupled to a chamber wall connected to the substrate device and configured to pull the cell sample toward the nanoelectrodes (not an embodiment of the invention). In this case, the chamber wall, as an actuator, is made of a material that contracts in response to external stimulation. The electrode device is connected to the substrate device via the chamber wall such that the nanoelectrodes are arranged in the space above the substrate surface, with the electrode tips arranged at a distance from the substrate surface and pointing toward the substrate surface. By contraction or mechanical compression of the chamber wall, the nanoelectrodes are moved toward the substrate surface until the electrode tips protrude into the cell sample on the substrate surface.This variant has the advantage that the nanoelectrodes can be adjusted using the actuator and inserted into the cell sample, which is fixed (adherently) arranged on the substrate device. By contracting or compressing the chamber wall, the penetration depth of the nanoelectrodes into the cell sample can be adjusted with increased precision and reproducibility. The movement can be limited by stop elements.

[0048] In a practical measurement procedure, cells such as enzymatically isolated cardiac muscle cells, enzymatically isolated neuronal cells, cardiac cells differentiated from stem cells, or neuronal cells differentiated from stem cells can be cultured on a nanoelectrode array. A first step can consist of coating the substrate surface (culture surface) with Matrigel or fibronectin, which improves adhesion and can also stimulate differentiation processes. Alternatively, the coating can be applied during manufacturing (using coated nanoelectrode arrays). After the cells are added, they adhere to the substrate surface, which can optionally be structured and have grooves or grooves with lateral anchor elements.

[0049] The penetration of the nanoelectrodes into the cell sample can be assisted by electroporation simultaneously with the actuation of the actuator. The diameter of the conductive tip is preferably less than or equal to 500 nm, in particular less than or equal to 200 nm, e.g., less than or equal to 100 nm, and / or conical, which enables a focused application of the membrane potential and facilitates penetration (breakdown) of the membrane.

[0050] In the electrophysiological measuring device according to all embodiments, the nanoelectrodes can carry an electrically insulating, viscous coating that can be stripped off the nanoelectrodes by the action of the cell membrane when the nanoelectrodes are introduced into the cell sample. The use of the viscous coating has the advantage of being relatively easy to apply and easily removed during use of the measuring device.

[0051] Further details and advantages of the invention are described below with reference to the accompanying drawings. They show schematically: Figures 1 to 6: Variants of electrophysiological measuring devices (not embodiments of the invention); Figure 7: An electrophysiological measuring device according to an embodiment of the invention; Figures 8 to 18: Further variants of electrophysiological measuring devices (not embodiments of the invention); Figure 19: An electrophysiological measuring device according to an embodiment of the invention; Figures 20 to 21: Further variants of electrophysiological measuring devices (not embodiments of the invention).

[0052] Embodiments of the invention are described below with exemplary reference to an electrophysiological measuring device equipped with up to twenty nanoelectrodes. The invention is not limited to these examples, but can be implemented accordingly with a measuring device or high-throughput testing device that has a larger number of nanoelectrodes arranged as a single row or as a two-dimensional array. For example, more than 500 nanoelectrodes (or up to 1000), each of which can be coupled to at least one measuring device, can be provided in a measuring device, so that a plurality of cell samples can be provided simultaneously and examined simultaneously or sequentially. A single nanoelectrode can also be provided.

[0053] Parts of the measuring device, referred to, for example, as a substrate device or an electrode device, can be implemented as separate or shared components. For example, the electrode device can at least partially form the substrate device, or the actuating device can at least partially form a chamber of the measuring device.

[0054] Furthermore, reference is made by way of example to measurements of ion currents and / or action potentials and / or patch-clamp measurements. For this purpose, suction openings provided in embodiments of the invention preferably have an adhesive surface coating on their edges to support the formation of the seal resistance. The invention is not limited to this application, but can be implemented accordingly with other electrophysiological measurements, such as the measurement of movements using electrical cell-substrate sensing (see, e.g., [18, 19]), or in combination with other measurements, such as fluorescence measurements or the detection of intracellular calcium or other ions.Details of electrophysiological measurements, such as the selection of electrical measurement parameters, the derivation of electrophysiological measurement signals from the nanoelectrodes, the arrangement of nanoelectrodes, the amplifier technology and / or the selection of perfusion media, details of sample preparation and details of the handling and, if applicable, cultivation of biological cell samples, such as the preparation of cell aggregates, are not described, as these are known per se from conventional techniques.

[0055] The invention is described in particular with respect to the configuration and operation of the actuating device and / or the configuration of a substrate device with a structured surface. The measuring device or a high-throughput testing device can comprise further components, such as liquid reservoirs, a fluidic device for supplying and removing media to the substrate, control elements (e.g., controllable valves), temperature control devices, humidification devices, and the like. A measuring device according to the invention can, in particular, be a stand-alone device for laboratory or industrial applications for examining cell samples. Alternatively, the measuring device can be part of a cultivation device for biological cells, which is designed in particular for cultivating cell aggregates. Furthermore, a control device can be provided with which the measuring device, in particular the electrode device and / or the actuating device, can be controlled.

[0056] In the described embodiments, the nanoelectrodes are preferably coated with an electrically insulating layer, e.g., SiO 2 , which leaves the electrode tips of the nanoelectrodes exposed, or completely coated with a viscous substance that exposes the conductive electrode tips of the nanoelectrodes upon penetration into the cell, without this being mentioned in the description of the individual figures. Furthermore, the cell culture surfaces can optionally be coated with Matrigel or fibronectin. Elastic materials can include, for example, PDMS or rubber.

[0057] Directions are referred to below in relation to the extent of the substrate (reference plane). Directions parallel to the reference plane are also referred to as lateral directions. The terms "top" and "bottom" and derived terms refer to the directions toward the cover and base sections of the measuring device, respectively. The drawings are schematic sectional views, some of which are supplemented by perspective views for clarity.

[0058] The drawings are schematic illustrations. In practice, the proportions and shapes of the parts of the measuring device may differ from those shown in the illustrations. For example, the chamber for receiving the cell sample may have a shape as described in

[16] .

[16] is incorporated by reference into the present disclosure, particularly with regard to the shapes of the chamber for receiving the cell sample.

[0059] Figure 1shows a first variant of an electrophysiological measuring device 100 with a substrate device 10 and an electrode device 20 (not an embodiment of the invention). The substrate device 10 comprises a substrate 11, which is provided for receiving the cell sample 1. The substrate 11 extends in a reference plane, which in Figure 1 is oriented substantially perpendicular to the plane of the drawing. Furthermore, the substrate device 10 is part of a chamber 90, the chamber wall 91 of which forms a vessel for receiving a culture medium 2 in the space above the substrate 11.

[0060] The electrode device 20 comprises a plurality of nanoelectrodes 21 arranged on a planar electrode carrier 22. The nanoelectrodes 21 extend in a longitudinal direction (parallel to the z-direction) perpendicular to the reference plane of the substrate 11. In this embodiment of the invention, the substrate 11 is formed by the electrode carrier 22 and an insulating layer 23 covering the electrode carrier 22. The insulating layer 23 leaves electrode tips 26 of the nanoelectrodes 21 exposed, thus establishing direct electrical contact between the nanoelectrodes 21 and the cytosol in the cell sample 1. The insulating layer 23, which forms a connection with the cell membrane 1A (lipid membrane), consists, for example, of SiO 2 with a thickness of 10 nm to 100 nm.

[0061] Alternatively, the nanoelectrodes can generally be formed from a non-conductive material, such as a plastic, and carry a conductive coating (10-100 nm) of a conductive material, e.g., gold. An insulating layer, e.g., of SiO 2 , with a thickness in the range of 10 nm to 100 nm can be provided on the conductive coating.

[0062] The nanoelectrodes 21 and mutually electrically insulated support sections 25 of the electrode support 22 are made of an electrically conductive material, such as gold, platinum, iridium, iridium oxide and rhodium oxide, titanide, or silver / silver chloride. The nanoelectrodes 21 have a conical or pyramidal shape with a longitudinal length of, for example, 100 nm to 10 µm and a diameter at the electrode tips of, for example, 50 nm to 200 nm. The electrode support 22 has insulation sections 24 that divide the electrode support 22 into the plurality of electrically insulated, electrically conductive support sections 25. Each of the support sections 25 is electrically connected to one of the nanoelectrodes 21. Furthermore, each support section 25 is connected to a measuring circuit 81 of a measuring device 80.The measuring device 80 further comprises reference electrodes 82, which are arranged in the chamber 90 above the substrate 11 and are electrically connected to the culture medium 2 in the chamber 90. The measuring circuits 81 are provided for measuring electrical voltage and / or current values ​​at the cell sample 1. The measuring device 80 is further optionally equipped with pulse generators for applying voltage pulses to the cell sample.

[0063] In the Figure 1In the electrophysiological measuring device 100 shown (not an embodiment of the invention), an actuating device for the relative movement of the cell sample 1 and the nanoelectrodes 21 is formed by a suction device 30, which comprises a suction opening 31 in the substrate device 10 and a pump device 32 (drive unit). With the pump device 32, comprising, for example, a microliter syringe (manufacturer Hamilton), the suction opening 31 can be subjected to a negative pressure, under the effect of which the culture medium 2 and with it the cell sample 1 are drawn to the suction opening 31. During the movement towards the nanoelectrodes 21, the cell membrane 1A is penetrated by cells of the cell sample 1, so that the electrode tips 26 protrude into the interior of the cells of the cell sample 1. Furthermore, the cells are electrically separated from the electrode carrier 22 by the insulating layer 23.

[0064] Although in Figure 1While only one suction opening 31 is shown, the realization of a measuring device is not limited to this variant. More suction openings 31 can be provided, distributed in the substrate 11 between the nanoelectrodes 21. All suction openings are connected to a common pumping device 32. Upon actuation of the pumping device 32, cells in several sections of the container formed by the chamber 90 are drawn evenly to the nanoelectrodes 21.

[0065] In a measurement method for the intracellular recording of at least one electrical measurement value on the biological cell sample 1, the following steps are carried out. First, a suspension of the culture medium 2 containing the cell sample 1 is filled into the chamber 90. The cell sample 1, which comprises, for example, heart cells differentiated from stem cells, can settle on the substrate 11 under the effect of gravity, although no or only negligible penetration of the cell membrane 1A by the nanoelectrodes 21 occurs. By actuating the pump device 32, the cell sample 1 is sucked onto the substrate 11, so that the nanoelectrodes 21 penetrate into the interior of the cells of the cell sample 1. A single nanoelectrode or multiple nanoelectrodes 21 can penetrate into individual cells. The number of nanoelectrodes 21 that have penetrated into each cell can be determined, for example, byby electrically measuring the resistance between adjacent nanoelectrodes 21. Subsequently, an electrophysiological measurement is performed in a conventional manner using the measuring device 80. The electrophysiological measurement can be performed on all nanoelectrodes 21 simultaneously or sequentially. A time dependence of measured values ​​can be recorded, e.g., to measure the propagation of an action potential from cell to cell in a cell sample 1.

[0066] After the measurement is completed, cell sample 1 can be removed from chamber 90. Alternatively, cell sample 1 can subsequently be cultured in chamber 90. For this purpose, additional medium, comprising, for example, nutrients and / or differentiation factors, can be added to the culture medium 2. After a predetermined culture time, at least one further electrical measurement can be performed on cell sample 1.

[0067] Figure 2 shows a modified variant of the electrophysiological measuring device 100 in different phases of the relative movement of the cell sample 1 and the nanoelectrodes 21 (not an embodiment of the invention). In this variant, the substrate 11 is formed by a substrate body 14, e.g., made of PDMS or polyvinyl chloride (PVC) with a thickness of 3 mm to 5 mm, with continuous suction openings 31, which extend in the z-direction perpendicular to the reference plane of the substrate 11. The suction openings 31 are, for example, round with a diameter, e.g., in the range of 1 to 4 µm. The chamber 90 is divided by the substrate 11 into an upper chamber section 92 and a lower chamber section 93, which are connected to one another via the suction openings 31. One of the suction openings 31 is shown with a schematically shown lateral suction line 33, which is connected to the pump device 32 (see Figure 2A). In practice, each of the suction openings 31 can be equipped with a lateral suction line 33 and / or at least one suction line can be connected to the lower chamber section 93. In addition, a perfusion line 33.1 can be provided at at least one of the suction openings 31, as shown schematically in Figure 2C is shown.

[0068] The suction opening 31 can optionally be covered with an ultra-thin elastic film (e.g. made of silicone), as in Figure 3 This offers the advantage that the diameter of the suction pores can be increased (in the range from 50 µm to 1 mm, as well as up to 10 mm), while preventing the cells from entering the suction opening. Further advantages of this embodiment will be described with reference to Figure 3 called.

[0069] The nanoelectrodes 21 of the electrode device 20 are arranged in a stationary manner in the lower chamber section 93. The nanoelectrodes 21 extend in the z-direction through the suction openings 31. The nanoelectrodes 21 are arranged countersunk in the suction openings 31. Preferably, the nanoelectrodes 21 are each positioned centrally in the suction openings 31. The electrode carrier 22 of the nanoelectrodes 21 is, as described above with reference to Figure 1 described, divided into individual carrier sections 25 by insulation sections 24. Each of the carrier sections 21 is electrically connected to a measuring circuit 81 of the measuring device 80.

[0070] When the suction line 33 is subjected to a negative pressure, the culture medium 2 is drawn, in particular, from the upper chamber section 92 through the suction openings 31. Together with the resulting fluid movement, individual cells or cell groups of the cell sample 1 are drawn to the suction openings 31. After the cell sample 1 has been sucked onto the suction opening 31, the cell sample 1 is partially drawn into the suction opening 31 if the suction line 33 is continued to be subjected to the negative pressure, as shown schematically in the Figures 2B and 2C As shown, the cell sample 1 strikes the electrode tip 26 of the nanoelectrode 21. Under the effect of the suction pressure, the cell sample 1 penetrates the nanoelectrode 21. Subsequently, electrical measurements are recorded on the cell sample 1.

[0071] The measuring device 100 according to Figure 2(not an embodiment of the invention) can be implemented in a modified manner according to one or more of the following features. Multiple nanoelectrodes 21 can be provided per suction opening 31. The suction openings 31 can have a different shape, e.g., as a groove in the substrate body. The suction openings 31 can be closed at the bottom in the substrate body and each equipped with a lateral suction line, wherein the nanoelectrodes are firmly embedded in the substrate body. Coatings of the nanoelectrodes and / or the culture surface formed by the surface of the substrate 11 can be provided.

[0072] The Figures 2A and 2B show an application of the measuring device for exclusive membrane potential measurements with a reference electrode 82 in the bath (cultivation medium 2). Figure 2Cshows a modified application of the measuring device for patch-clamp measurements with a fixed cell ("cell attached" measurement) with a ground electrode 82.1 in liquid contact with the lower chamber section 93.

[0073] Particularly for patch-clamp measurements, it may be advantageous to provide adhesion sections 14.1 at the upper end of the suction openings 31 on the substrate 11, facing the upper chamber section 92. The adhesion sections 14.1 (shown schematically hatched) each surround the upper edge of a suction opening 31. Adhesion sections 14.1 may also be provided in the variants according to the Figures 2A and 2B (not embodiments of the invention) and may be provided in the embodiments of the invention.

[0074] The adhesion sections 14.1 form a high-resistance contact (> 100 megohms) or a gigaseal at the edge of the suction openings 31. This allows the cell-attached measurement to be performed on the cell membrane surrounding the nanoelectrode 21 with respect to the membrane potential measured with the nanoelectrode 21. Both lines for potential (V) and membrane current (Im) are isolated from each other and can be measured simultaneously with the measuring device 80, which comprises a patch-clamp amplifier 83.

[0075] In Figure 2C The penetration of the cell 1 is shown after applying a negative pressure by means of the suction line 33 and / or the lower chamber section 92, whereby the high-resistance contact or Gigaseal contact to the substrate 11 is formed. Figure 2A The reference electrode shown is not required.

[0076] The at least one nanoelectrode 21 is centrally positioned within the suction opening 31, which is also referred to as a perfusion pore. The suction opening 31 simultaneously forms a perfusable compartment around the respective at least one nanoelectrode 21. The ground electrode 82.1 is arranged in this compartment, electrically insulated from the nanoelectrode 21 to form an additional virtual ground. The ground electrode 82.1 is also used for cell-attached patch-clamp measurements.

[0077] Figure 2C illustrates a further advantageous feature of a measuring device (not an embodiment of the invention) which, in the variant of the Figure 2Cand can also be optionally provided in the described embodiments. The nanoelectrode 21 has a tip distance A from the surface of the substrate (distance of the electrode tip in the longitudinal direction of the nanoelectrode 21 from the surface, see arrow). The tip distance A is preferably selected in the range from 100 nm to 5 µm. The diameter of the suction opening 31 is preferably selected in the range from 1.5 µm to 5 µm for measurements on single cells and in the range from 10 µm to 100 µm, in particular 10 µm to 20 µm, for measurements on cell aggregates.

[0078] The Figure 3 The variant of the electrophysiological measuring device 100 shown is similar to the measuring device 100 according to Figure 1constructed (not an embodiment of the invention). The substrate device 10, together with the chamber 90, forms a vessel for receiving the cultivation medium 2 containing the cell sample 1. The substrate device 10 is equipped with a flexible, deformable support membrane 12, which is intended to receive the cell sample 1 and optionally for the growth of the cells of the cell sample 1. The support membrane 12 divides the chamber 90 into an upper chamber section 92 and a lower chamber section 93. The support membrane 12 is, for example, a silicone membrane, for example with a thickness in the range of 1 to 5 µm. The support membrane advantageously also acts as an insulator by tightly enclosing the shaft of the nanoelectrode. This enables the optional use of nanoelectrodes not coated with an insulating layer 23. The shaft of the nanoelectrode can be extended for this application, e.g., to up to 50 µm.

[0079] The electrode device 20 is formed by stationary nanoelectrodes 21 in conjunction with a planar electrode carrier 22 at the bottom of the chamber 90. The electrode device 20 forms a bottom part with which the chamber 90 is closed at the bottom. The carrier membrane 12 is stretched between the walls 91 of the chamber 90 so that it extends parallel to the extension of the electrode carrier 22. The carrier membrane 12 can be arranged at a distance from the electrode tips 26 of the nanoelectrodes 21 or can touch them.

[0080] The actuating device for the relative movement of the cell sample 1 and the nanoelectrodes 21 comprises the suction device 30 with at least one suction opening 31 in the bottom part of the substrate 11 and a pump device 32 connected to the suction opening 31. When the suction opening 31 is subjected to a negative pressure, liquid is sucked out of the lower chamber section 93, so that the carrier membrane 12 with the cell sample 1 is drawn to the electrode device 20 and in particular to the nanoelectrodes 21. The nanoelectrodes 21 pierce the carrier membrane 12 and the cell membrane 1A of the cells of the cell sample 1 located on the carrier membrane 12 until the electrode tips 26 are positioned inside the cells. The desired electrophysiological measurement then takes place.

[0081] The support membrane 12 is provided by a separating device which separates the space for receiving the cell sample 1 (upper chamber section 92) from the nanoelectrodes 21 in the lower chamber section 93 as long as the suction device 30 is not actuated and the support membrane 12 is not pulled towards the nanoelectrodes 21.

[0082] The measuring device 100 according to the Figures 1 to 3 can be manufactured from plastic, metallic components and / or ceramics using available manufacturing processes in fluidic microsystem technology.

[0083] At the Figure 4In the variant shown, the substrate device 10 of the electrophysiological measuring device 100 is formed by the mouth region 13A of a suction tube device (not an embodiment of the invention). The suction tube device is, for example, a measuring pipette 13 (partially shown) which is designed for a patch-clamp measurement (patch-clamp pipette). The edge of the mouth region 13A (pipette tip) at the end of the measuring pipette 13 preferably has a thickness d in the range of, for example, 1 µm to 10 µm. The edge of the mouth region 13A has an adhesive surface coating to support the formation of the sealing resistance. The inner diameter D of the mouth region 13A is in the range of, for example, B. 1 µm to 200 µm are selected, whereby the small inner diameters (up to e.g. 30 µm) are used for measurements on single cells and D > 10 µm for cell-attached patch-clamp measurements on cell aggregates.The electrode device 20 comprises two or more nanoelectrodes 21 (one of the electrodes is shown in cross-section for illustrative purposes) arranged protrudingly on the peripheral edge of the mouth region 13A. The longitudinal directions of the nanoelectrodes 21 extend parallel to the axial direction of the measuring pipette 13.

[0084] Connecting lines 27 run from the nanoelectrodes 21 to measuring circuits 81 of a measuring device 80. The connecting lines 27 are mounted outside the orifice, for example on the outer surface of the measuring pipette 13, and are electrically insulated relative to the environment ( Figures 4A and 4B ) or free-standing ( Figure 4C ). The measuring device 80 additionally comprises a reference electrode 82, which is arranged inside the measuring pipette, and a measuring amplifier 83, which is designed for measuring ion currents.

[0085] In the embodiment of the invention, the adjusting device for the relative movement of the cell sample 1 ( Figure 4B, 4C and 4D ) and the nanoelectrodes 21 a suction device 30. This is in the variant according to Figure 4 by the outlet opening 34 of the suction tube device (orifice of the measuring pipette) and a pump device connected to the suction tube device (piston of the pipette, not shown). The outlet region 13A forms the suction opening, at the edge of which the nanoelectrodes 21 are arranged.

[0086] The nanoelectrodes 21 may carry an insulating layer 23 covering the nanoelectrodes 21, leaving the electrode tips 26 uncoated ( Figure 4BThe electrode tips 26 protrude, for example, approximately 100 nm to 5 µm from the insulating layer 23. The insulating layer 23 can improve contact with the cell membrane, which is advantageous for both single cells and cell aggregates. The insulating layer 23, e.g., made of SiO 2 , is intended for orifices 34 with an inner diameter, e.g., in the range of 10 to 100 µm, and for measurements on cell aggregates connected via nexus connections.

[0087] No insulation layer 23 is required, for example, if so-called "cell-attached" measurements are carried out on individual cells and a high-resistance contact (> 100 megaohms) or a gigaseal is formed at the edge of the mouth area 13A ( Figures 4C and 4D ). This would be the case, for example, for an inner diameter D of the orifice 34 in the range of 1 to 30 µm, a thickness in the range of 2 to 5 µm and the suction of a single cell.

[0088] When performing the electrophysiological measurement procedure with the electrophysiological measuring device 100 according to Figure 4 (not an embodiment of the invention) a cell suspension containing the cells or cell aggregates to be examined is provided in a vessel (not shown). The measuring pipette is inserted into the cell suspension. When the measuring pipette is subjected to negative pressure, a cell or cell aggregate is sucked to the mouth area 13A (cell-attached configuration), whereby the cell is moved to the nanoelectrodes 21 until these penetrate into the cell interior and establish electrical contact with the cytosol in the cell. At the same time, the membrane patch 1B is formed (see Figures 4B, 4C). Electrophysiological measurements, such as patch-clamp measurements, are performed in a conventional manner. In multicellular samples, membrane potentials are measured on different cells, which, for example, in myocardial cell samples, are connected to each other by nexus contacts. Here, multiple potentials from different cells can be measured and used as reference values ​​for cell-attached measurements.

[0089] A special advantage of the Figure 4The advantage of the measuring device shown is that, due to the positioning of the nanoelectrodes 21 at the mouth area 13A, no additional reference electrode needs to be contained in the vessel containing the cell suspension. The measuring pipette 13 can be freely handled. This is particularly advantageous when changing solutions for testing substances or the like. Furthermore, the intracellular potential (V) can be directly tapped and used as a reference value for the patch-clamp measurement. In the cell-attached configuration, ion currents through the insulating membrane section and, simultaneously, the action potentials generated by the cell aggregate or the individual cell can be measured.

[0090] At the Figure 4B In the variant shown (not an embodiment of the invention), patch-clamp measurements can be performed without creating a Gigaseal isolation of the membrane patch and the cell membrane 1A. With further details, Figure 4BA schematic representation of the current flow, starting from the "virtual ground" (IV converter, black dot in pipette) through the membrane patch of the cell membrane 1A, the nanoelectrode 21, an outer conductive (e.g., gold) metal jacket 13B surrounding the measuring pipette 13, and then to the voltage generator 84, which generates a voltage clamp signal. In contrast to conventional patch-clamp technology, the bath solution is not connected to the measuring device 80 (patch-clamp amplifier).

[0091] In a modified version ( Figure 4C, not an embodiment of the invention), such measurements can be carried out under conditions of gigaseal isolation (hatched areas on the pipette tip) of the membrane patch. In this case, external insulation of the nanoelectrode 21 with an insulating layer 23 (e.g., SiO 2 ) is not required. The membrane patch is insulated from the remaining cell membrane 1A and bath solution by contact with the pipette tip. In this variant, the voltage clamp signal is applied via the nanoelectrode (R NE ) to the virtual ground, with the gigaseal resistor (RGig) isolating the membrane patch from the bath solution.

[0092] In another modified version ( Figure 4D, not an embodiment of the invention), the pipette is provided on the outside with two mutually insulated conductor tracks that run from the measuring device 80 directly to the pipette tip (patch pore). Two nanoelectrodes 21.1, 21.2 are provided on the pipette tip, which contact the (-) input and the output of an operational amplifier of the measuring circuit 81 for 2-electrode voltage clamp via the two conductor tracks. The nanoelectrode 21.1 is used to measure the cell membrane potential of the cell sample 1 in the form of a cell aggregate, and a feedback current is injected via the nanoelectrode 21.2 to keep the membrane potential constant. The bath solution is not connected to a separate ground potential. The measuring device 80 further comprises the measuring amplifier 83 for measuring ion currents, in particular for a patch-clamp current measurement, which is connected to a reference electrode 82 inside the pipette.

[0093] Since the myocardial cells of the cell aggregate are connected to each other by nexus contacts, the potential of the cells drawn into the patch pore is kept constant. The ion currents at the "clamped" membrane patch are measured using the current-voltage converter. The inner diameter of the pipette tip is between 10 and 200 µm.

[0094] The production of Figure 4 The variant of the measuring device shown (not an embodiment of the invention) comprises a multiple coating of the glass surface of the measuring pipette 13 with a layer sequence comprising a sputtered, structured gold layer on the glass surface with a thickness in the range of 100 nm to 500 nm for forming the conductor tracks, and a SiO 2 layer thereon with a thickness in the range of 50 nm to 500 nm. The nanoelectrodes 21.1, 21.2 are arranged, for example, by being placed on the gold layer at the outlet opening.

[0095] The Figures 5 to 6 illustrate further modifications of the electrophysiological measuring device 100 (not embodiments of the invention) and Figure 7 illustrates an embodiment of the electrophysiological measuring device 100 according to the invention), each with a substrate device 10, an electrode device 20 comprising an electrode carrier 22 and nanoelectrodes 21, and a suction device 30 in a schematic sectional view ( Figures 5 , 7 ) or schematic plan view ( Figure 6 ). Above the substrate device 10, a chamber 90 with a conical shape narrowing towards the substrate 11 is provided. The chamber 90 is designed to accommodate a culture medium 2 and the cell sample 1 in the form of a cell aggregate (see Figure 5, not an embodiment of the invention). Further variants of this embodiment are described below with reference to the Figures 19(embodiment of the invention) and 20 to 21 (not embodiments of the invention).

[0096] The chamber wall of chamber 90 has according to Figure 5 an upper, flatter wall section 91A and a lower, steeper wall section 91B (not an embodiment of the invention). This subdivision can have advantages when fixing the cell sample 1 in the chamber 90. The lower, steeper section 91B can be provided with an inner, electrically conductive coating, which forms a reference electrode 85 for an electrophysiological measurement or serves for impedance measurements to detect movements of the cell aggregates.

[0097] The substrate device 10 comprises a substrate 11 which, as shown in Figure 1 by the electrode carrier 22 with electrically conductive carrier sections 25 and insulation sections 24 (see Figure 6, not an embodiment of the invention). The insulation sections 24 divide the electrode carrier 22 into the carrier sections 25, each of which is connected to a nanoelectrode 21 and a measuring circuit 81 of the measuring device 80. The nanoelectrodes 21 have a height in the range of, for example, 1 to 10 µm. The measuring device 80 comprises the measuring circuits 81, which are each connected to the nanoelectrodes 21, and a measuring amplifier 83, which is designed for measuring ion currents, in particular a patch-clamp current measurement, and is connected to a reference electrode 82.

[0098] The suction device 30 comprises a central suction opening 31 in the substrate 11, which is connected to a pumping device (not shown) for applying a negative pressure. Additionally, a ring opening 34 is provided, which surrounds the nanoelectrodes 21 in the reference plane of the substrate 11. The ring opening 34 can be connected to the same pumping device or, as in Figure 5As shown, the electrode device 20 can be connected to a separate pumping device via a flow channel 35 below the electrode device 20. The outer ring opening 34 serves to suck in the cell sample 1 and fix it in the funnel of the chamber 90. In a modified variant, the ring opening 34 can be replaced by several suction openings, which are evenly distributed in a circle around the central suction opening 31.

[0099] The embodiment of the invention according to Figure 7 is constructed similarly to the electrophysiological measuring device 100 according to Figure 5. In contrast to this, no ring opening is provided in the substrate 11. Furthermore, the chamber wall 91 forms a projection 91C extending circumferentially and radially inwardly at a distance from the substrate 11. The projection 91C forms an undercut, which additionally supports the fixation of the cell sample 1 on the substrate 11 with the nanoelectrodes 21. Below the projection 91C, a concentric suction channel is formed, which is provided with at least one flushing line 95. The cell sample 1 can be sucked into the chamber via this suction channel, which enables the fixation of the cell sample 1 as an alternative to the ring opening 34 according to Figure 5 and also enables perfusion of cell sample 1 with liquids, e.g. solutions of test substances.

[0100] When using the electrophysiological measuring device 100 according to the Figures 5 to 6(not embodiments of the invention) and 7 (embodiment of the invention), a cell aggregate forming the cell sample 1 is first pipetted into a culture medium 2 in the chamber 90. When the suction opening 31 and / or the ring opening 34 and / or the flushing line 95 is subjected to a negative pressure, the cell sample 1 is drawn towards the bottom of the chamber 90 onto the nanoelectrodes 21 until these penetrate the cells of the cell sample 1. At the same time, the cell sample 1 in the variant according to Figure 5 from the lower, steeper wall section 91B of the chamber wall. Since the cells of cell sample 1 are connected to each other via nexus contacts, the measuring device 80 can record electrical measured values ​​that represent the entire cell sample 1 (see

[16] ).

[0101] With the measuring devices according to the Figures 5 to 6(not embodiments of the invention) and 7 (embodiment of the invention), several functions can advantageously be performed. With the suction opening 31 in the center and the optionally provided concentric ring opening 34 or the concentric suction channel in the lower funnel section, the cell sample is sucked in, causing penetration of the membrane. Furthermore, the suction opening 31 is preferably surrounded by a sealing ring section 28, which forms an electrical resistance as in conventional patch-clamp measurements. The sealing ring section 28 is formed by the surface of an insulating section 24.

[0102] In another electrophysiological measuring device, the actuating device is formed by an electrode drive device 40, as shown schematically by way of example in the Figures 8 to 11is shown (not embodiments of the invention). The electrode drive device 40 comprises a mechanical drive that acts on the electrode device 20, in particular the electrode carrier 22 and / or the nanoelectrodes 21. In the illustrated variants, the electrode drive device 40 comprises an electrically stimulated piezo actuator made of a piezoelectric material, such as piezoelectric ceramics or piezoelectric crystals. Alternatively, other materials can be used that change their geometric properties in response to other, e.g., optical, external stimulation.

[0103] In these variants (not embodiments of the invention), the electrode carrier 22 with the nanoelectrodes 21 is arranged to be movable relative to the chamber wall 91 of the chamber 90, wherein a seal 91D is preferably provided between the electrode device 20 and the chamber wall 91, which seal allows a sliding movement of the electrode carrier 22 and is simultaneously liquid-tight.

[0104] According to a first variant of the measuring device (see Figure 8 , 10 and 11, not embodiments of the invention), the substrate device 10 comprises a substrate 11 having a substrate body 14 and / or a porous membrane 16, each containing through-going electrode openings 15. The electrode carrier 22 of the electrode device 20 is arranged on the underside of the substrate device 10. Nanoelectrodes 21 of the electrode device 20 protrude into the electrode openings 15 toward the chamber 90. Upon actuation of the electrode drive device 40, the nanoelectrodes 21 are advanced through the electrode openings 15.

[0105] In the variant according to Figure 8(not an embodiment of the invention) the substrate body 14 is made of a compressible material, such as PVC with a thickness of 100 nm to 100 µm. On the upper side of the substrate body 14 facing away from the electrode device 20, the porous membrane 16 is arranged. The porous membrane 16 is made of an inelastic material, such as PDMS or PVC with a thickness of 100 to 500 µm and is attached internally to the chamber wall 91. For clarity, Figure 8 The pore membrane 16 is shown at a distance from the substrate body 14. In reality, the pore membrane 16, which extends parallel to the reference plane of the substrate 11 with the substrate body 14, touches the upper surface of the substrate body 14. The pore membrane 16 represents a solid support for the cell sample 1 (Figure 8A).

[0106] When the electrode drive device 40 is actuated, the electrode device 20 presses against the substrate body 14, compressing it between the electrode device 20 and the pore membrane 16, while the nanoelectrodes 21 are advanced through the electrode openings 15 until the electrode tips 26 penetrate the cell sample 1 (Figure 8B). In this state, the desired electrical parameters of the cell sample 1 are measured with the measuring device 80.

[0107] According to an alternative variant, the at least one electrode opening 15 of the substrate device 10 is closed with a cover membrane 14A and the electrode drive device 40 is configured to move the nanoelectrodes 21 through the cover membrane 14A into the cell sample 1 ( Figures 9A and 9B , not embodiments of the invention). As in Figure 8The electrode carrier 22 with the nanoelectrodes 21 is arranged to be movable relative to the chamber wall 91 of the chamber 90, whereby the liquid-tight seal 91D allows the sliding movement of the electrode carrier 22. The cover membrane 14A forms the substrate surface for receiving the cell sample 1, and it is like the pore membrane 16 ( Figure 8 ) is made of an inelastic material and is attached to the inside of the chamber wall 91. The cover membrane 14A is provided by a separating device which separates the space for receiving the cell sample 1 (interior of the chamber 90) from the nanoelectrodes 21 as long as the electrode drive device 40 is not actuated and the nanoelectrodes 21 are not advanced.

[0108] According to a further alternative variant of the invention, the substrate 11 of the substrate device 10 comprises a substrate body 14 with a closed surface, wherein in this case nanoelectrodes 21 of the electrode device 20 are arranged at a distance from the substrate body 14 and protrude from the chamber 90 to the substrate body 14 (top-down configuration, see Figures 9C and 9D , not embodiments of the invention). The electrode device 20 is connected to the chamber wall 91 and is displaceable perpendicular to the reference plane of the substrate 11. Upon actuation of the electrode drive device 40, the nanoelectrodes 21 are advanced toward the substrate 11 until the electrode tips 26 penetrate the cell sample 1, so that the desired electrical quantities of the cell sample 1 can be measured with the measuring device 80.

[0109] When assembling according to the Figures 9C and 9D(not embodiments of the invention) the substrate body 14 may comprise a transparent plate, e.g. made of plastic or glass, so that a visual and / or optical-microscopic observation of the cell sample 1 through the substrate body 14 is possible.

[0110] Optionally, the substrate body 14 is coated with a cell-compatible material, such as Magel, poly-L-lysine, gelatin, or fibronectin. A lateral flushing line 95 (perfusion channel), also optionally provided, serves to supply cells. Furthermore, the flushing line 95 allows the generation of a negative pressure, which assists the advancement of the electrode device 20.

[0111] A spacer 91E (elastic spacer element, shown hatched), e.g. made of PDMS or rubber, is provided between the electrode carrier 25 and the substrate body 14, which seals the chamber and is also equipped with at least one stop element 91F (not an embodiment of the invention). The spacer 91E can be provided at the edge of the chamber 90 (as shown) and optionally additionally in the volume of the chamber, surrounding the nanoelectrodes 21. Spacers 91E can thus be placed not only at the edge of the chamber 90, but also between the nanoelectrodes 21 and the substrate body 14. This enables simultaneous movement of the nanoelectrodes 21 into multiple cells on a substrate surface without the electrode carrier 25 being bent by the movement.The movement of the nanoelectrodes 21 can be restricted by the at least one stop element 91F (limiting element), which enables a defined penetration depth of the nanoelectrodes 21 into the cytosol to be set.

[0112] In the variant according to the Figures 9C and 9D(not an embodiment of the invention), the solid SiO 2 coating of the nanoelectrodes 21 can be omitted. Instead, a coating 23A of the nanoelectrodes 21 with a thin layer (e.g., 10 nm to 100 nm or more, e.g., up to 1 µm) of a viscous material, for example Vaseline, mixtures of silicone oil and Parafilm, polydimethylsiloxane (PDMS), gelatin, each with admixtures of extracellular matrix (Magel, BioCoat, fibronectin, poly-L-lysine or amino acids) and nutrients, is preferably provided. Upon introduction of the nanoelectrodes 21 into the cell sample 1, the coating 23A is partially stripped off the nanoelectrodes 21, so that contact with the cytosol is established (see Figure 9D , not embodiments of the invention). The coating 23A of the nanoelectrodes 21 with the viscous material can also be provided in the other embodiments of the invention.

[0113] According to a further alternative variant, the substrate of the substrate device 10 comprises a substrate body 14 with electrode openings 15 made of a non-compressible material ( Figures 10, 11 , not embodiments of the invention). In this case, the electrode device 20 is as in Figure 8 arranged below the substrate body 14, with the nanoelectrodes 21 projecting into the electrode openings 15. Deviating from Figure 8 The electrode device 20 is arranged at a distance z 0 from the substrate body 14. The distance z 0 is selected such that the nanoelectrodes 21 can be moved a sufficient feed path through the electrode openings 15 into the cell sample 1 ( Figures 10A / B). The distance z 0 is, for example, 100 nm to 3 mm. The underside of the substrate body 14 forms a stop for the electrode device, so that a defined penetration depth of the nanoelectrodes 21 in the cell sample can be set. In addition, the substrate body or the cell array can be equipped with flexible spacers (spacer elements) and stop elements (see Figures 9C, 9D , not embodiments of the invention).

[0114] In addition, the substrate body 14 can be provided on its underside with a guide cone 14B, which interacts with suitably shaped conical sections 22A of the electrode carrier 22 ( Figure 10C, not an embodiment of the invention). This advantageously supports the centered guidance of the nanoelectrodes 21 through the electrode openings 15. The guide cone 14B can be made of a flexible material (e.g., PDMS), which has a sealing and insulating effect when the nanoelectrode moves. In this case, an insulating coating of the nanoelectrode can optionally be omitted ( Figure 10D , not an embodiment of the invention).

[0115] Figure 11(not an embodiment of the invention) shows a structured surface of the substrate body 14 with substrate projections 17, which have laterally projecting anchor elements 18. The substrate projections 17 with the anchor elements 18 fix the cell sample 1 during the piercing of the nanoelectrode 21 when this is pushed forward through the electrode opening 15 with the electrode drive device 40. The substrate projections 17 have an elongated wall shape, so that a groove (channel) for receiving the cell sample 1 is formed between the substrate projections 17. The cell sample 1 can, for example, form a fiber growing in the longitudinal direction of the groove, which fibers are fixed in the channel by anchor elements. The upper surface of 17 is provided with nanostructures that prevent cell adhesion, and the groove can optionally be provided with adhesion-promoting coatings. The nanoelectrodes 21 and the electrode openings 15 are, for example, B. arranged in a row, as shown in the top view in the right part of Figure 11 The peripheral edges of the electrode openings 15 each form sealing surfaces for supporting the cell membrane of the cell sample during the measurement. Further details and functions of the structured surface are described below with reference to the Figures 15 and 17 (not embodiments of the invention) are described.

[0116] In a further electrophysiological measuring device (not an embodiment of the invention), the actuating device is formed by a substrate drive device 50, as shown schematically by way of example in Figure 12 The substrate drive device 50 comprises a mechanical drive that acts on the substrate device 10, in particular the substrate body 14 with the electrode openings 15. The electrode device 20 with the nanoelectrodes 21 and the electrode carrier 22, which, for example, as shown in Figure 1is fixedly arranged at the bottom of the chamber 90. The substrate drive device 50 is formed by the substrate body 14 itself, which is made, for example, of a material that can be stimulated electrically, optically, magnetically, or ultrasonically. In response to external stimulation, e.g., a change in an electrical voltage applied to the substrate body 14, the substrate body 14 contracts.

[0117] The thickness of the substrate body 14 decreases ( Figures 12A / 12B ) until the nanoelectrodes 21 protrude above the upper surface of the substrate body 14 and penetrate into the cell sample 1. The substrate body 14 preferably has a cell-compatible coating, e.g., made of SiO 2 , on its upper side, which forms the substrate surface for receiving the cell sample.

[0118] In a further electrophysiological measuring device (not an embodiment of the invention), the actuating device is formed by a magnetic drive device 60, as shown schematically by way of example in Figure 13 The magnetic drive device 60 comprises magnetic particles 61 coupled to the cell sample 1 and a main magnet 62 arranged below the electrode device 20. The magnetic particles 61 are, for example, glass-coated micro-iron spheres or magnetic nanoparticles. The main magnet 62 is a permanent magnet or a switchable electromagnet. The main magnet 62 is designed to attract the cell sample 1 toward the nanoelectrodes 21 upon magnetic interaction with the magnetic particles 61 until they penetrate into the cells of the cell sample 1.

[0119] The cell sample 1 can be moved in a direction perpendicular to the reference plane of the substrate 11 and optionally also laterally along the reference plane of the substrate 11. For the lateral movement, a displacement of the main magnet 62 parallel to the reference plane of the substrate 11 can be provided. Details of the movement of the cell sample 1 can be realized, for example, as described in

[17] .

[0120] In a further electrophysiological measuring device (not an embodiment of the invention), the actuating device is formed by a chamber drive device 70, which is provided on a chamber wall 91 connected to the substrate device 10 and is designed to move the nanoelectrodes 21 towards the cell sample 1 (see Figures 14 to 16). In the illustrated variants, the chamber drive device 70 is formed by the chamber wall 91 itself. Alternatively, the chamber drive device 70 may be a separate component connected to the chamber wall 91, e.g., made of an elastically deformable material, or another part of the chamber 90, and deforming the chamber wall 91 upon actuation (see, e.g., Figure 16 ).

[0121] This variant is realized as a top-down configuration, in which the electrode device 20 with the nanoelectrodes 21 is arranged at a distance above the stationary substrate 11 of the substrate device 10. In the top-down configuration, a separating device is provided by the chamber wall 91, which separates the cell sample 1 inside the chamber 90 from the nanoelectrodes 21 as long as the chamber drive device 70 is not actuated and the nanoelectrodes 21 are not advanced ( Figures 14A , 15A and 16 ).

[0122] According to Figure 14A (not an embodiment of the invention) The chamber 90 of the electrophysiological measuring device 100 is bounded at the bottom by the substrate 11, e.g., made of glass, laterally by the chamber wall 91, and at the top by a cover part 94, e.g., made of glass, into which the electrode device 20 and a feed funnel 96 are integrated. The electrode device 20 comprises an electrode carrier 22, which is divided by insulation sections 24 into carrier sections 25, each with a nanoelectrode 21, as described above. Each nanoelectrode 21 is connected to a measuring circuit 81 of the measuring device 80. The chamber wall 91 consists of a piezoelectric material that contracts when an electrical voltage changes. Alternatively, the chamber wall can consist of an elastic material that can be compressed by a chamber drive device.

[0123] To perform an electrophysiological measurement, the measuring device 100 is filled via the feed funnel 96 with a cell suspension comprising the cell sample 1 and a culture medium 2. The internal dimensions and the inner surface of the feed funnel 96 and the chamber 90 can be formed such that the supply of the cell suspension is supported by capillary forces. Advantageously, the cell sample 1 adheres to the surface of the substrate 11 before the nanoelectrodes 21 move. The surface of the substrate 11 can, as described with reference to Figure 17 described, to promote adhesion (nanostructuring as an adhesion aid). After the cell suspension has been added, the nanoelectrodes 21 are at a distance from the cells of the cell sample 1 ( Figure 14A ).

[0124] Upon electrical stimulation of the chamber drive device 70, ie the chamber wall 91, the electrode device 20 is lowered until the nanoelectrodes 21 penetrate the cells of the cell sample 1 ( Figure 14B ). In this state, at least one electrical measurement value is recorded on cell sample 1.

[0125] The chamber 90 of the electrophysiological measuring device 100 may be a closed vessel with a cylindrical interior ( Figures 14A and 14B , not an embodiment of the invention). Alternatively, the chamber 90 may have a cuboid-shaped interior and in particular be formed as a flow system (channel), as in the plan view in Figure 14C The nanoelectrodes 21 in the cover part 94 can, for example, form an electrode array, particularly for measurements on cell layers (monolayers). Figure 14C further illustrates that the chamber 90 may be laterally sealed by a spacer 91E (not an embodiment of the invention).

[0126] The Figures 15A and 15B show a modification of the electrophysiological measuring device 100 according to Figure 14, wherein the substrate 11 has a structured surface with substrate protrusions 17 and lateral anchor elements 18 (not embodiments of the invention). The substrate protrusions 17 form straight grooves at the bottom of the chamber 90, in which the cell sample 1 is adherently positioned. The grooves have, for example, a width of 10 µm to 300 µm and a depth of 3 µm to 300 µm. The lateral anchor elements 18 have, for example, an extension parallel to the reference plane of the substrate of 100 nm to 100 µm. Adherent growth exclusively or predominantly in the grooves can be supported by an adhesion-promoting coating in the grooves. While cells without the anchor elements 18 may only adhere superficially in the grooves and detach from the substrate 11, particularly during the formation of contracting cardiac muscle fibers (see

[14] ,

[15] ), the anchor elements 18 provide additional fixation of the cells.

[0127] Figure 15Bshows a top view of a variant of the electrophysiological measuring device 100, in which the nanoelectrodes 21 form two straight rows that run along the alignment of the grooves formed by the substrate protrusions 17 (not an embodiment of the invention). A measuring circuit 81 is provided at each of the nanoelectrodes 21 (illustrated only at one of the nanoelectrodes 21). At the opposite ends of each row, a stimulation electrode 97A, 97B is provided for stimulation and excitation propagation in one direction along the row.

[0128] In the variant according to Figure 15 The chamber's side accesses are open. In this variant, the chamber is placed, for example, in a cell culture vessel and then placed in a laterally sealed perfusion device so that the chamber can be perfused along the recesses in the base (grooves with and without anchor elements). The open variant of the Figure 15ensures access of larger volumes of culture medium in a cell culture vessel to the cells by diffusion. The volume is in the very flat closed version Fig. 14C limited by the low height.

[0129] The variants in particular of the Figures 15A and 15B (not embodiments of the invention) further have the particular advantage of enabling intracellular measurements of the propagation of an action potential across a cell culture, e.g., a fiber structure. This was known with conventional techniques only with extracellular electrodes. The propagation of the action potential can be measured, in particular, as a function of the effect of a perfusion fluid. The perfusion fluid can contain, for example, biologically active substances, in particular drugs, whose effect on the cell sample 1 is to be recorded by the electrophysiological measurement.

[0130] In Figure 16A modified variant of the chamber drive device 70 is shown, which comprises a holder 71 and a lever actuator 72 (not an embodiment of the invention). The chamber 90 with the substrate device 10 and the electrode device 20 is fixed in the holder 71. The cover part 94 with the electrode device 20 is arranged to be movable relative to the chamber wall 91. The lever actuator 72 acts on the cover part 94. The chamber wall 91 is made of an elastic (compressible) material. Upon actuation of the chamber drive device 70, the cover part 94 is displaced downward by the lever actuator 72, so that all nanoelectrodes 21 are simultaneously moved towards the cell sample 1 and penetrate the cells. The lever actuator 72 comprises, for example, a piezoelectric material or, alternatively, a combination of two materials with different thermal expansion coefficients, e.g., a bi-metal composite.The bi-metal composite has the advantage of a larger feed path (e.g. up to 100 µm or more) compared to a piezoelectric material (e.g. up to 2 to 5 µm).

[0131] As a modification of the variant in Figure 16 the actuator 72 can be part of the electrode carrier 22 (not an embodiment of the invention).

[0132] For fixing a cell sample 1, in particular single cells and / or cell aggregates, to the substrate of an electrophysiological measuring device, e.g., on the floor in a perfusion gap, substrate projections 17 with lateral anchoring elements 18 can be provided, as shown in Figures 17A and 17B (not embodiments of the invention). The substrate projections 17 with lateral anchoring elements 18 can be provided in all embodiments of the invention described here that are equipped with an adjusting device. Alternatively, the substrate projections 17 with lateral anchoring elements 18 can be provided in measuring devices that do not have an adjusting device and in which, therefore, no targeted relative movement of the cell sample and nanoelectrodes is provided (not embodiments of the invention). Furthermore, the substrate projections 17 with lateral anchoring elements 18 can be provided in substrates, e.g.,in the form of cover glasses with a structured surface, in which no nanoelectrodes are provided (not embodiments of the invention). The production of the substrate protrusions with the anchor elements is carried out, for example, using nanoimprint lithography technology.

[0133] The improved fixation of the cell sample is advantageous because cells with poor adhesion could, for example, be flushed out of the chamber when shear forces occur in a perfusion fluid due to the solution flow.

[0134] The Figures 17A and 17B(not embodiments of the invention) show substrate protrusions 17 in columnar or wall form, which at their upper ends each have lateral anchor elements 18 one above the other (so-called double or triple T-profiles). Alternatively, the substrate protrusions 17 can form, for example, simple T-profiles or have more than three lateral anchor elements 18 one above the other. The double T-profiles are characterized by a spacing X between the substrate protrusions 17, which is selected for the fixation of individual cells, e.g., in the range of 300 nm to 10 µm, and for the fixation of cell aggregates and fibers, e.g., in the range of 30 µm to 1 mm.Furthermore, the double-T profiles are characterized by a height Z of the substrate projections 17 in the range of 500 nm to 1 mm, a width of the anchor elements x 1 in the range of 100 nm to 1 mm, an undercut depth of the anchor elements x 2 in the range of 100 nm to 100 µm, a thickness of the anchor elements z 1 in the range of 100 nm to 500 nm and a distance of the anchor elements z 2 in the range of 50 nm to 10 µm.

[0135] In the case of multiple T-profiles, the anchor elements can have different extensions parallel to the reference plane of the substrate, e.g. increasing towards the substrate (see Figure 17B ). For example, dimension X3 is between 2% and 50% larger than X2; with an additional anchor element, dimension X4 could be 5-50% larger than X3.

[0136] The top surface of the T-structures has a lower adhesion for cells than the other surfaces, which results in preferential adhesion and growth of the cells at the bottom along the depression (groove). A nub 17.1, for example, to prevent adhesion is shown as an example in Figure 17B shown (not an embodiment of the invention).

[0137] The arrays for fibers in T-structures enable the measurement of action potentials (APs) at various locations along the fibers, thus allowing the determination of propagation velocity or intercellular resistance. Advantageously, the measurement is performed on a structure aligned with the grooves, which facilitates these measurements due to the simplified geometry—compared to undirected growth in a monolayer.

[0138] Alternatively or additionally, different geometric orientations, tapering of the grooves, a circular course (2 fibers meet) of the grooves formed between the substrate projections can be realized.

[0139] The substrate protrusions 17 can form elongated nano-grooves, which, for example, form adhesion elements for filopodia of individual cells or cell aggregates. Between the substrate protrusions 17, openings can be provided in the substrate, in which electrodes can be moved (see, for example, Figure 11 , not an embodiment of the invention). Alternatively, recesses between substrate projections 17, e.g., with T-structures, can have other geometric shapes in addition to a straight alignment (growth of simple longitudinal fiber directions), such as cross-shaped, circular, and / or tapered (not an embodiment of the invention).

[0140] Figure 18shows a variant of the electrophysiological measuring device 100, in which the substrate device 10, the electrode device 20 and parts, in particular the actuators, of the actuating device form a flexible measuring probe 110 (not an embodiment of the invention). Figure 18Only the distal end of the measuring probe 110 is shown, which furthermore has a flexible shaft for insertion, e.g., through an endoscope into tissue, and is equipped with at least one drive unit for controlling the actuators of the actuating device. The measuring probe 110 can be implemented with all of the above-described variants of actuating devices, with reference here to a suction device 30 as an example. Upon actuation of the actuating device 30, the cell sample 1 in the form of the tissue is drawn to the nanoelectrodes 21, so that these penetrate the cell sample 1. The measuring probe 110 is constructed, for example, from flexible polymer materials and metals. Furthermore, the measuring probe 110 can be equipped with all of the above-described variants of measuring devices 80, in particular measuring circuits, pulse generators, reference electrodes, ground electrodes, and / or amplifiers.

[0141] The measuring probe 110 for measuring membrane potentials and / or ion currents can be used for the detection of intracellular membrane potentials of individual neuronal cells and / or the measurement of transmembrane ion currents, e.g., in brain tissue under in vivo conditions. The use of such probes and their placement in brain tissue is described, for example, in

[20] . A disadvantage of conventional probes is that only the surface potentials of groups of neurons are detected, but not the intracellular potentials of individual cells or the currents through cell membranes. The measuring probe 110 enables new applications of electrophysiological measuring devices and an extension of the method described in

[20] .

[0142] According to Figure 18AThe measuring probe 110 is equipped with a plurality of measuring units (e.g. measuring units for intracellular measurements of membrane potentials or current measurements through membranes in the cell-attached configuration), which are essentially like the variants of the electrophysiological measuring device described above, in particular as in the Figures 1 , 3 , 4 , 8and / or 9 (not embodiments of the invention). In a probe body 111 of the measuring probe 110, which forms the substrate device 10, e.g., has a diameter of 1 to 3 mm and is made of a flexible polymer, cylindrical depressions are embedded, each equipped with a nanoelectrode 21 and a suction opening 31. The nanoelectrodes 21 are connected to the measuring device 80 via electrical conductors. The suction openings 31 are each connected to a pump device (not shown) via a suction line. Each suction line can be equipped with a valve for selectively controlling individual measuring units. The depressions can be used for the measurement of intracellular membrane potentials with an ultra-thin membrane (in Figure 18A not shown) must be closed (see Figure 3). For further details of the units of measurement, reference is made to the variants described above. Alternatively or in addition to the Figure 18A Other adjustment devices may be provided for the suction devices shown (see e.g. Figures 8 and 9 , not embodiments of the invention).

[0143] The measuring probe 110 is inserted through an endoscope or other hollow tube into the tissue to be examined. When the pumping device is activated, the tissue is drawn toward the measuring probe 110, allowing the nanoelectrodes 21 to penetrate the cells. Applying a negative pressure causes the nerve tissue (or a membrane with the adhered nerve tissue) to bulge into the cylindrical recess, leading to the penetration of individual cells (optionally through the membrane) with the nanoelectrodes 21.

[0144] In order to improve the derivation of intracellular potentials and / or ion currents, the substrate device 10 and the electrode device 20 of the measuring probe 110 can be adapted as shown in the Figures 18B and 18C shown (not an embodiment of the invention). At the suction openings 31 in the recesses of the probe body, the latter is equipped on its surface with annular attachments 31.1, which in their dimensions (inner diameter e.g. 10 µm to 200 µm) correspond to the Figures 4A to 4DThe annular attachments 31.1 (corresponding to the pipette tips of the embodiments described above) are made, for example, of glass or SiO 2 or a polymer, such as PDMS, and form a high-resistance contact or gigaseal contact with the accommodated cell. The annular attachments 31.1 are located in the cylindrical recesses, which are connected to one or more pumping devices by thin tubes (capillaries). In the variant according to Figure 18A Ring-shaped attachments 31.1 (not shown) can also be provided, which surround the edges of the recesses on the surface of the probe body (not an embodiment of the invention).

[0145] In Figure 18BFeatures of a further variant of the measuring probe 110 are shown, in which the annular attachments 31.1 are equipped with at least one nanoelectrode 21 in the edge region (not an embodiment of the invention). In this case, the negative pressure is applied within the annular attachment 31.1, which leads to the creation of a seal with the surface of the nerve tissue and simultaneously causes the penetration of cells with the nanoelectrodes.

[0146] Figure 18C (not an embodiment of the invention) shows a larger section of the measuring probe 110 in a schematic top view with multiple measuring units for measuring membrane potentials (top row) or ion currents with the cell-attached configuration and membrane potentials using nanoelectrodes (bottom row). The lines to the measuring device 80 are routed within the measuring probe 110. The suction lines or other drive elements for moving the nanoelectrodes are not shown.

[0147] The Figure 19 illustrates a further variant of the embodiment of the electrophysiological measuring device 100 according to the invention. Figures 20 to 21 Illustrate variants of the electrophysiological measuring device 100 (not embodiments of the invention). In these figures, the measuring device 100 comprises, in particular, a substrate device 10, an electrode device 20 with a nanoelectrode 21, a suction device 30, a measuring device 80 with a ground electrode 82.1, and a chamber 90 for receiving a culture medium 2 and the cell sample 1 in the form of a cell aggregate in a schematic sectional view.

[0148] According to Figure 19 is instead of the Figure 5An annular gap 34A is provided in the chamber wall 91 at the lower end of the chamber 90, which forms an annular suction channel. The annular gap 34A has a larger outer diameter than the diameter of the lower end of the chamber 90. The chamber wall 91 thereby receives a projection 91C for further fixation of the cell sample 1 (see also Figure 7 ). The annular gap 34A is connected to a pumping device (not shown), with which the annular gap 34A can be subjected to a negative pressure relative to the pressure in the chamber 90. In a modified variant, the annular gap 34A can be replaced by several suction openings, which are evenly distributed in the chamber wall.

[0149] When the annular gap 34A is subjected to negative pressure, the cell sample 1 is advantageously sucked in laterally (radially outward and simultaneously toward the bottom of the chamber 90). Due to the symmetrical arrangement of the annular gap 34A, this exerts a force toward the nanoelectrode 21 at the bottom of the chamber 90. Although the nanoelectrode is arranged at an angle of 90 degrees relative to the suction direction in the annular gap 34A, the nanoelectrode 21 reliably penetrates the cell sample 1.

[0150] The nanoelectrode 21 is arranged at the bottom of the chamber 90, laterally adjacent to the annular gap 34A and laterally adjacent to the additional perfusion opening provided in the center. A cell membrane of the cell sample is thus supported by the cell carrier 10 in the immediate vicinity of the nanoelectrode 21. This advantageously stabilizes the cell membrane.

[0151] The perfusion opening located centrally at the bottom of the chamber 90 is connected to a flushing line 95, which provides a perfusion channel for perfusing the cell sample 1 and / or a connection to a reference electrode 82 of the measuring device 80. The flushing line 95 is formed, for example, as a microfluidic channel in the cell carrier 10.

[0152] Figure 20 (not an embodiment of the invention) shows a variant of the measuring device 100, in which the chamber 90 deviates from the conical shape, e.g. according to Figure 19 at its lower end has the shape of a spherical shell section 91D. This shape has the advantage that an undesired deformation of the cell sample 1, e.g. by a projection 91C (see Figure 19) or by inner edges on the bottom of the chamber 90, so that injuries are avoided, especially to sensitive cell samples. The spherical shell section 91D is preferably dimensioned such that approximately one third to one half of the sucked-in cell sample 1 sits in the spherical shell section 91D. Cell samples 1 in the form of cell aggregates have, for example, a diameter in the range of 300 µm to 1 mm, so that the diameter D of the chamber 90 at the upper end of the spherical shell section 91D and the height of the spherical shell section 91D in the axial direction is preferably selected in the range of 500 µm to 800 µm.

[0153] To move the cell sample 1 to the nanoelectrode 21 at the bottom of the chamber 90, an annular opening 34 is provided, which is connected to a pumping device (not shown) via a flow channel 35 below the electrode device 20. When subjected to a negative pressure, the annular opening 34 serves to suck in the cell sample 1 and fix it in the chamber 90.

[0154] According to Figure 21 (not an embodiment of the invention), a cylindrical chamber 90 can be provided, in the bottom of which the annular opening 34 for aspirating the cell sample 1 is arranged. Furthermore, an annular bead 91D is provided at the bottom of the chamber 91, which protrudes upward in the axial direction and laterally surrounds the annular opening. Advantageously, the annular bead 91D forms a positioning and aspiration aid when securing the cell sample 1.

Claims

1. Electrophysiological measuring apparatus (100) which is configured for intracellular detection of at least one electrical measuring value on a biological cell sample (1), comprising - a substrate device (10) configured to receive the cell sample (1), - an electrode device (20) with at least one nanoelectrode (21) which extends in a longitudinal direction in each case and can be introduced intracellularly into the cell sample (1) which is received by the substrate device (10), and - an actuating device which is configured for moving of the at least one nanoelectrode (21) and / or the cell sample (1) relative to one another and along the longitudinal direction of the at least one nanoelectrode (21) such that the at least one nanoelectrode (21) penetrates into the cell sample (1), wherein - the actuating device comprises a suction device (30) configured to draw the cell sample (1) towards the at least one nanoelectrode (21), - the suction device (30) comprises at least one suction opening (31, 34) in the substrate device (10) and a pump device (32) which is configured to apply a negative pressure to the at least one suction opening (31, 34), and - the at least one suction opening (31, 34) is shaped such that when the at least one suction opening (31, 34) is subjected to the negative pressure, the cell sample (1) is moved along the longitudinal direction to the at least one nanoelectrode (21), characterized in that - a chamber (90) with a chamber wall (91) having a conical shape which narrows towards the substrate (11) is provided above the substrate device (10), wherein the chamber (90) is configured to receive a cultivation medium (2) and the cell sample (1) in the form of a cell aggregate, and - a projection (91C) for fixing the cell sample (1) is provided above the substrate device (10) in the chamber wall (91) at the lower end of the chamber (90).

2. Electrophysiological measuring apparatus according to claim 1, wherein - the at least one suction opening (31, 34) is each provided with an adhesion portion (28) configured to form a high-impedance contact or a gigaseal contact with a received cell sample (1).

3. Electrophysiological measuring apparatus according to any one of claims 1 to 2, wherein - the at least one nanoelectrode (21) is arranged protruding on the substrate device (10), and - the at least one suction opening (31, 34) is arranged immediately adjacent to the at least one nanoelectrode (21).

4. Electrophysiological measuring apparatus according to claim 3, wherein - the electrode device (20) comprises a plurality of nanoelectrodes (21), and - the at least one suction opening (31) is arranged between the nanoelectrodes (21).

5. Electrophysiological measuring apparatus according to one of the preceding claims, wherein - the at least one suction opening comprises a ring opening (34) which is arranged in the substrate device (10) and surrounds the at least one nanoelectrode (21) along a lateral extension of the substrate device (10), wherein the ring opening (34) is configured for suctioning the cell sample (1) and fixing it to the substrate device (10).

6. Electrophysiological measuring apparatus according to one of the preceding claims, wherein - the at least one nanoelectrode (21) is arranged in the at least one suction opening (31) in the substrate device (10).

7. Electrophysiological measuring apparatus according to claim 6, wherein - the at least one nanoelectrode (21) extends parallel to an inner wall of the suction opening (31, 33, 34) in the substrate device (10).

8. Electrophysiological measuring apparatus according to one of claims 6 to 7, wherein - the at least one nanoelectrode (21) is arranged such that its free end comprises a predetermined distance from the surface of the substrate device (10).

9. Electrophysiological measuring method for intracellular detection of at least one electrical measuring value on a biological cell sample (1), comprising the steps of - receiving the cell sample (1) on a substrate device (10), - arranging at least one nanoelectrode (21) of an electrode device (20) such that it projects intracellularly into the cell sample (1) which is received by the substrate device (10), and - measuring the at least one electrical measuring value on the cell sample (1), wherein - a movement of the at least one nanoelectrode (21) and / or the cell sample (1) relative to one another and along the longitudinal direction of the nanoelectrodes (21) is provided with an actuating device (30-70) such that the at least one nanoelectrode (21) penetrates into the cell sample (1), wherein - the actuating device comprises a suction device (30) which is configured for drawing the cell sample (1) to the nanoelectrodes (21), wherein the suction device (30) comprises at least one suction opening (31, 33, 34) in the substrate device (10) and a pumping device which is configured for applying a negative pressure to the at least one suction opening (31, 33, 34), and - when the at least one suction opening (31, 33, 34) is subjected to the negative pressure, the cell sample (1) is moved along the longitudinal direction to the at least one nanoelectrode (21), characterized in that - a chamber (90) with a chamber wall (91) having a conical shape which narrows towards the substrate (11) is provided above the substrate device (10), wherein a cultivation medium (2) and the cell sample (1) in the form of a cell aggregate are accommodated in the chamber (90), wherein - a projection (91C) is provided above the substrate device (10) in the chamber wall (91) at the lower end of the chamber (90), with which projection the cell sample (1) is fixed.