WO2015188911A1

WO2015188911A1 - Electrophysiological measuring arrangement and electrophysiological measuring method

Info

Publication number: WO2015188911A1
Application number: PCT/EP2015/000979
Authority: WO
Inventors: Philipp Julian KÖSTER; Oliver Klink; Carsten Tautorat; Uwe Scheffler; Thomas Kröger; Denise Franz
Original assignee: Universität Rostock
Priority date: 2014-06-10
Filing date: 2015-05-12
Publication date: 2015-12-17
Also published as: DE102014008606B3

Abstract

The present invention relates to an electrophysiological measuring arrangement (100) and an electrophysiological measuring method, in which a first carrier (12-1) is provided, comprising an aperture region (10), which is embodied in the region of the first carrier (12-1) for controlled sealing attachment of a biological object (O) and which, to this end, is embodied with at least one aperture (14) and with a wall region (11), which surrounds the aperture (14) in a forming manner. Furthermore, a second carrier (12-2) is provided, which is embodied on at least one side (12-2a) assigned to the first carrier (12-1) for adhesive attachment and/or cultivation of biological objects (O) to be examined, wherein the first and the second carrier (12-1, 12-2) are embodied in a controllable manner and in a manner approachable to one another with the side (12-2a) of the second carrier (12-2) assigned to the first carrier (12-1) substantially facing the first carrier (12-1) and the aperture region (10) of the first carrier (12-1).

Description

Electrophysiological measuring arrangement and electrophysiological measuring method

FIELD OF THE INVENTION

The present invention relates to an electrophysiological measuring arrangement as well as to an electrophysiological measuring method using the electrophysiological measuring arrangement according to the invention.

BACKGROUND OF THE INVENTION Various methods and devices are known, which are used in the field of electrophysiology to biological objects - especially cells in the broadest sense, cell organelles, oocytes and their fragments - with regard to embedded in respective membranes and / or there attached proteins and their transport properties, whereby vesicles, liposomes or other more or less artificial systems can also be used.

Frequently, electrical currents and / or electrical voltages are impressed and / or measured between a measuring electrode and a counterelectrode, which are arranged between the biological object. The measured currents and / or voltages should provide information about the underlying physiological processes, in particular transport processes, conformational changes and the like. Frequently, comparatively very low signal strengths are present. For achieving a suitable signal-to-noise ratio, therefore, high sealing resistances, that is to say the lowest possible residual electrical conductivities, are necessary over the membrane itself or in the contact or contact region between the membrane of the biological object to be examined and the aperture wall.

Often in this context also the task arises to investigate single cells or an ensemble of cells in a cell assembly or even to feed the cell assembly as a whole to a measuring method, e.g. To capture interactions between directly or indirectly neighboring cells.

In a naturally chosen environment, such as a sample taken or in an uncontrolled cell ensemble, the difficulty is that the metrological devices must be based on the random geometry of the then found cell ensemble. This often represents a considerable effort and may also involve difficulties in reproducibility of different measurements in relation to each other.

It would therefore be desirable if a corresponding cell ensemble were provided in a well-defined and predictable manner, possibly also with a predictable design, or else a randomly arranged cell ensemble or e.g. a section of an organ or tissue could be reliably attached to a wearer and subjected to an electrophysiological measurement process.

SUMMARY OF THE INVENTION

The object of the invention is therefore to provide an electrophysiological measuring arrangement and a corresponding measuring method using the electrophysiological measuring arrangement in which measuring devices and biological objects to be examined are set in a predictable and controlled manner with particular reliability and reproducibility in relation to each other can be aligned.

The object underlying the invention is achieved in an electrophysiological measuring arrangement according to the invention with the features of independent patent claim 1. Furthermore, the object underlying the invention in an electrophysiological measurement method according to the invention with the features of independent claim 10 is achieved. Advantageous developments are described in the dependent claims. On the one hand, the subject of the present invention is therefore an electrophysiological measuring arrangement which has a first carrier and a second carrier. The first carrier is formed with an aperture region, which is formed in the region of the first carrier for the controlled sealing attachment of a biological object or a plurality of biological objects and has a corresponding structure and which is formed with at least one aperture or opening and with a wall portion which surrounds the aperture or opening. The second carrier is formed at least on a side associated with the first carrier for adherent attachment and / or cultivation of biological objects to be examined. It can also be it can be seen that cuts of tissues or organs are applied or formed on the wearer to form the biological objects to be examined in this way. The measuring arrangement is embodied such that the first carrier and the second carrier can be controlled and, with the side of the second carrier associated with the first carrier, approach the first carrier and the aperture region of the first carrier substantially facing one another.

The task of the first carrier is therefore to provide at least one aperture, e.g. in the sense of a measuring opening or measuring capillary, which then serve for the sealing or "sealing" attachment and thus for the formation of a so-called gigaseal.

The second support serves to provide an adhesion region or cultivation region, e.g. in the manner of a culture medium on which one or preferably several cells or other biological objects arrange, train or even cultivate. Thus, e.g. be thought that the second carrier is initially in a culture medium and there is overgrown after appropriate inoculation with a lawn to be examined biological objects, in which case the area, which is later assigned and approximated the first carrier, in whole or in part by the biological Objects, eg Cells or the like is covered.

Alternatively or additionally, it may also be provided that cuts of tissues or organs are applied or formed on the carrier in order to provide the biological objects to be examined in this way.

The measures make it possible according to the invention

(A) measurement not only on a biological object to be examined, e.g. a cell, but to perform

(B) measurements on several similar or different biological objects to be examined, e.g. Cells perform out of an ensemble

(C) to select an especially suitable individual from an ensemble, eg with simultaneous microscopic examination, (D) also to investigate interactions between indirectly and immediately adjacent biological objects to be examined or

(E) even simultaneously or sequentially consider several biological objects and their direct or indirect interaction.

For this purpose, it is advantageous that the first and second carrier are approachable in a controlled manner, thereby to approach a respective biological object of the or aperture as a measuring aperture, until a sealing attaching the biological object to be examined with its membrane at the Aperture results, eg in the sense of a gigaseal and, where appropriate, by assisting with further measures via the measuring aperture, e.g. by applying pressure, by applying electrical charge in the wall region which forms the aperture e.g. via an otherwise electrically isolated from the measuring medium control electrode assembly or also using surface modifications on the wall region for controlling the attachment, e.g. in the sense of a doping, a certain surface texture or chemical modifications of the surface. By approximating the first and second supports in relation to each other in a controlled manner, it is also possible to reduce the total volume of remaining free measuring space, which is generally e.g. filled by an aqueous solvent or measuring medium, to be kept particularly small in order thereby to be able to change the measuring environment quickly, e.g. by rapid perfusion and in so-called concentration jump experiments or also to be able to change the experimental conditions for the biological objects to be examined as quickly as possible.

In a preferred embodiment of the electrophysiological measuring arrangement, at least one actuator is formed for the controlled approach of the first carrier and the second carrier to each other. Through this, the first carrier and / or the second carrier can be moved relative to each other controlled.

Alternatively or additionally, means for self-organization, self-leveling, and / or self-adjustment in the approach and / or alignment, in particular positioning marks or webs, contact or pressure sensors or the like may be formed. Provision may be made for the first carrier to be moved and / or aligned with the second carrier resting thereon or for the second carrier to be moved and / or aligned with the first carrier resting thereon. It is also possible for both carriers to be moved independently of one another, possibly forming a plurality of actuators.

In another preferred embodiment of the electrophysiological measuring arrangement according to the present invention, it is provided that the respective actuator is designed to linearly displace the first carrier and / or the second carrier along one or more of the three spatial directions x, y, z. Additionally or alternatively, a respective actuator for rotating the first carrier and / or the second carrier may be formed around one or more of the three spatial directions x, y, corresponding to axes of rotation X, Y, Z.

Tilting of the substrate can also be done by self-leveling the substrate because e.g. PDMS carriers are particularly soft and flexible as a substrate. Actuators are also conceivable which cover all or part of these possibilities of movement shapes of the first and second carriers in relation to each other. However, it may also be formed a corresponding plurality of actuators covering one or more of these possibilities of movement of the first and second carrier with respect to each other.

As a result of these measures, alignment of the first carrier and the second carrier with respect to one another can take place in a particularly flexible manner. Thus, it is also possible in a particularly flexible and reliable manner to align and to measure the biological objects to be measured - and possibly randomly distributed on the second carrier - with respect to the measuring structures, in particular with respect to one or more apertures orientate. In this case, not only the distance of the biological objects to be examined to the respective aperture can be controlled, but the orientation of biological objects to be examined and assigned aperture in all three spatial directions and axes of rotation with respect to each other.

It is particularly advantageous if the second carrier, at least on the side associated with the first carrier, that is to say the side, which in an experiment or an electrophysiological measuring method then the side provided with the aperture or area of the first carrier is approximated, is formed with or from a material which controls the growth, adhesion and / or culturing of the biological objects to be examined, in particular promotes. For this purpose, it may be provided that the side of the second carrier associated with the first carrier is formed with or made of a suitable material, for example with or made of a soft and / or flexible material, in particular of PDMS or the like. The adhering, growing or cultivating material may also be provided or formed in a structured manner on the surface, for example to promote a particular geometry in adhering, growing and / or cultivating an ensemble of biological objects to be examined, eg in lattice form or the like ,

For the purposes of the invention, biological objects which are subjected to an examination may be cells, cell organelles, oocytes, bacteria or their combinations or fragments, in each case in the broadest sense. Artificial or partially artificial essentially biological structures are also conceivable, for example in the form of vesicles, liposomes, micelles, membrane fragments or the like, in which proteins are incorporated and / or deposited in a natural or artificial manner.

The objects to be examined may generally be natural or partially or completely artificial biological objects. Also, non-biological objects are assayable to produce e.g. to investigate pure lipid structures and their modifications. In the following, only biological objects are discussed, but all variations described in this sense are to be included as measuring objects.

In another embodiment of the measuring arrangement according to the invention, the aperture region is formed in the region of a first carrier which has an upper side and a lower side. In this case, a respective wall region forming an aperture with respect to the upper side and / or with respect to the underside of the first carrier may project partially or completely.

The first carrier to be provided may also be referred to as a first base, a first substrate or a first base substrate. The provision of such a first substrate or of such a first carrier mechanically stabilizes the measuring arrangement and in particular the arrangement of the biological object to be examined as examined, and allows a macroscopic subdivision of the measuring arrangement with respect to the electrolyte bath to be subdivided in the sense of subdividing one Measuring cuvette or wet cell in compartments with measuring and counter electrode.

A respective wall region forming an aperture can also be integrated in the inner wall of the hole in combination with the electrode arrangement.

On the basis of the first substrate or the first carrier, one or more apertures may be formed with corresponding wall areas with respect to the top outstanding or emphasizing.

Alternatively, these may also be everted flush inwardly at the top to protrude on the underside of the first carrier or substrate; However, this is not mandatory and can be omitted with appropriate thickness of the membrane.

The AusmajS the respective Eindülpens or protrusion affects the inner wall of the respective wall area and thus the interaction surface with the interaction of the membrane of the biological object. Moreover, the choice of the degree of protrusion or indentation makes it possible to adapt to the particular measurement objects available, for example with regard to their shape or number in the measurement solution.

The first carrier may be formed as - in particular planar - plate element with front or top and back or bottom. Other geometries are conceivable.

Alternatively, it is possible to deviate from the plate shape, in which, for example, the shape of a pipette, eg in the sense of a classic patch pipette is taken up. The wall region which forms an aperture can be designed in the manner of a lateral surface or as a combination of lateral surfaces. In each case, the shell of a cylinder, a prism, a truncated cone and / or a truncated pyramid can be gripped back, with a corresponding wall thickness. With regard to the shape of the wall region for forming the aperture, there are therefore numerous possibilities. These can be selected depending on the shape and the other - eg mechanical, geometric and / or electrical - properties of the biological objects to be examined.

An aperture forming wall portion may be formed with or from a material of the group of materials including glass, quartz glass, silicon, carbon, and combinations thereof. With regard to the choice of material, the properties of the underlying biological objects can also be taken into account, for example with regard to the surface structure or surface charge of the membrane exterior and / or the membrane interior of the biological objects, for example, to provide particularly intimate adhesion and thus further supporting the increase in the sealing resistance of the seal.

The diameter of the aperture and in particular the inner diameter of one or the aperture forming wall portion may have a value in the range of about 0 μιη to about 50 μιη, preferably in the range in the range of about 1 μπι to about 50 μπι. One or the wall forming an aperture may have a height or depth in the range of about 0 μm to about 20 μm over the top or bottom of the carrier or substrate.

The stated dimensions with regard to height and depth of the wall regions and their diameters can be further based on the geometric conditions of the biological objects to be examined, in particular their size and the mechanical properties of their membranes, and consequently values other than those specifically indicated.

To form a measuring circuit, a measuring electrode can be provided in the region or within the aperture or in a region on the rear or underside of the carrier or substrate.

There may be a counter electrode outside the aperture and in the area on the front or top of the carrier.

The measuring electrode and the counterelectrode are preferably arranged on opposite sides of the carrier or substrate or the measuring aperture, in any case in such a way that when a preferably biological object is to be measured, this is arranged between the electrodes and, when a suitable electrode is formed. th sealing resistance or seals this electrically separated, preferably with a very high resistance, ideally greater than 1 GO.

The basic structure of the electrophysiological measuring arrangement according to the invention according to this embodiment thus includes in particular the provision of a measuring electrode arrangement and a counter electrode arrangement, between which an electric current and / or an electrical voltage can be measured, wherein between the measuring electrode arrangement and the counter electrode arrangement just the biological object to be measured In the region of the aperture and its wall region, the residual conductivity, ie the conductivity between the membrane of the biological object and the wall region of the aperture, is as small as possible, so that the actually measured electrical conductivity is as low as possible due to the sealing or sealing attachment Currents and / or electrical voltages can be considered to be caused by the properties of the membrane of the biological object, for example by transport processes, charge shifts within or over across the membrane, through substrate binding or dispensing, or the like.

According to a further aspect of the present invention, an electrophysiological measuring arrangement is proposed, in which the aperture region of the first carrier has a plurality of apertures, in particular in a grid or matrix-like arrangement and / or analogous organotypic forms, e.g. of the hippocampus or other biological structures, and in which the second carrier on the side associated with the first carrier one of the arrangement of the plurality of apertures corresponding arrangement of position structures and in particular of positioning recesses and / or projections for receiving and / or fixing a biological Object, wherein in particular directly adjacent positioning structures or positioning recesses / projections are each connected by a connecting structure and in particular a channel and / or a web on or in the first carrier associated side.

Such a structure for the second carrier gives the possibility of forming or arranging an ensemble of biological objects to be examined with a well-defined arrangement and topology which is particularly accessible to a measurement. According to a further aspect of the present invention, an electrophysiological measuring method is defined, in particular using the electrophysiological measuring arrangement according to the invention.

This method includes but is not limited to the following steps:

(1) providing the first carrier and the second carrier in a fluid measuring medium,

(2) providing the second support with adhered or suspended biological objects on the second support, optionally by cultivating the biological objects on the second support,

(3) Positioning and aligning the first and second carrier to each other, in particular in a controlled manner, and thereby - in particular controlled - approaching the first and second carrier to each other such that thereby at least one biological object is approximated to the region of at least one aperture, so that a contact between the membrane of the biological object and the aperture can take place or takes place at least directly,

(4) suction and suction of the membrane of the biological object located in the region of the aperture by means of negative pressure across the aperture,

(5) thereby attracting the sucked and sucked portions of the membrane and sealingly attaching the sucked and sucked portions of the membrane to the inner wall of the aperture and

(6) performing an electrophysiological measurement on the biological object located in the region of the aperture.

In a preferred embodiment of the method according to the invention, a randomly distributed cell ensemble or network on the second carrier is examined by means of the controlled positioning and alignment of the first and second carriers by means of an electrophysiological measurement.

In another embodiment of the method according to the invention, the approximation of the first and second carriers in relation to each other results in controlled manner and Welse means of the controlled positioning and alignment of the first and second carrier reduces the total existing volume of a free measuring space, namely compared to a procedure without controlled positioning and aligning the first and second carrier with respect to each other, in particular thereby to a measurement environment of rapidly changing biological object, in particular by rapid perfusion and / or concentration jump experiments, and / or to quickly change the experimental conditions for the biological objects to be examined. In a further embodiment of the method according to the invention, an approximation and / or positioning and in particular a tilting of the first and / or the second carrier as a substrate via a self-leveling of the respective carrier or substrate, especially if their respective material soft and / or flexible is.

The self-leveling could be done by means of a joint between the punch and the sinking unit, ie the second carrier and an actuator, by a flexible material, such as e.g. PDM or others, and / or

(C) by small and the distance defining and holding feet on the stamp and on the opposite side on the substrate, ie between the first and the second carrier.

With the latter measure, it is thus also possible to define the distance between the chip and the substrate and / or to set a perfusion speed.

These and other aspects of the present invention will be further clarified on the basis of the accompanying drawings.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-C show a schematic and sectional side view of a

Embodiment of the electrophysiological measuring device according to the invention in various intermediate states in their use. 2A shows in plan view an embodiment of a first carrier having a matrix arrangement of a plurality of apertures. 11 shows in plan view an embodiment of a second carrier with an ensemble of adherent cells as biological objects arranged in a matrix form corresponding to the matrix form of FIG. 2A. shows in plan the superposition of the first and second carrier of FIGS. 2A and 2B at or after approaching each other. shows in sectional side view another embodiment of the electrophysiological measuring arrangement according to the invention, in which the wall portion of an aperture rises above the top of an underlying support. show in schematic and sectional plan view different cross-sectional shapes of an aperture and the respective underlying wall portion. show in a similar manner as in Fig. 1A in a schematic and sectional side view of various embodiments of an electrophysiological measuring device according to the present invention, in which a respective aperture are formed by wall portions which correspond to lateral surfaces of different geometric body. show in schematic and sectional side view various aspects of the use of the electrophysiological measuring device according to the invention. show in schematic and sectional side view details in the attachment of a biological object to a respective embodiment of the electrophysiological measuring arrangement.

FIG. 12 shows graphs showing measurement results in single-cell measurements using the electrophysiological techniques of the present invention Measuring arrangement and / or the electrophysiological logging method according to the invention.

FIG. 13 shows measurement results in so-called single channel measurements using the electrophysiological according to the invention

Measuring arrangement and / or the electrophysiological measuring method according to the invention.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Embodiments of the present invention will be described below in detail. All embodiments of the invention and also their technical features and properties can be isolated individually and combined randomly and combined with each other arbitrarily and without restriction.

In the following, unless stated otherwise, structurally and / or functionally identical, similar or identically acting features or elements in connection with the figures are designated by the same reference numerals. Not every case of their occurrence repeats a detailed description of these features or elements.

First of all, drawings are generally referred to. In electrophysiology, among other things, the patch-clamp technology is applied to e.g. Perform ion channel analyzes for drug testing. In the manual patch-clamp method and its refinements, e.g. on single cells - electrical currents and voltages are measured which - e.g. of ion channels - are caused in the membranes of biological cells.

Due to the growing importance of electrophysiological examinations and the time and effort involved in their implementation, a great demand has arisen for automated electrophysiological measurement techniques and in particular for planar patch clamp and other automated patch clamp or APC systems.

The functionalities of the manual patch-clamp procedure and APC systems are fundamentally the same. The problem with both types of systems is the necessity of forming a high-resistance sealing resistor between the measurement object O, for example a cell O, and the measuring arrangement 100. One also speaks of the need for a so-called gigaseal, that is to say a sealing resistance in the gigaohm range. Thus, the electrical insulation - eg the cell interior ZelläujSeren - z. As described by a patch pipette or measuring aperture, as shown in a flat cell array, such as in an APC scheme in Figs. 9A to 9E, 10 and 1 1.

By means of a measuring aperture 14, a cell may be aspirated as a biological object O. A small, limited part of the cell membrane M, which is referred to as a patch, is thereby drawn in with a slight negative pressure. As a result, in the cell-attached measurement according to FIG. 9B, the measuring surface or, in the case of a whole-cell measurement according to FIG. 9D, the cell interior is electrically sealed in the mega to gigaohm region towards the cell outer. The cell-attached configuration also allows current measurement on individual ion channels in the cell membrane.

The gigaseal rate and the sealing resistance of the manual patch clamp and the APC systems are a measure of the quality of the possible ion channel measurements. To date, a 100% gigaseal rate is not possible. The training of the Gigaseal depends on many factors that are not accessible to active influence.

With the manual patch clamp method, only a sufficiently high gigaseal rate can be achieved with freshly produced glass pipettes. When developing chips for APC systems, attention must be paid to reducing the surface roughness and avoiding sharp edges. Furthermore, a careful combination of the intracellular and extracellular buffers can lead to an improvement of the gigaseal. However, this has only limited possibilities of control and influence due to the need to use physiological buffers.

The present invention can also be understood as an analysis system which consists of an adherent cell culture platform and has a chip-based analysis hardware in the sense of a patch-clamp chip, wherein one, in particular elongated. Patch pipette with improved perfusion capability is provided. The present invention can be used in particular as a system for drug tests in basic research as well as in drug discovery in adherent and / or crosslinked cells. When carrying out cells on a cell culture platform in terms of eg lowerable punch, which can also be referred to as a second carrier in the context of the invention, cultivated and then perpendicular to the so-called patch clamp chip, which is also the first carrier in the context of the invention can be referred to lowered. Optionally, by means of further suction of the cell membranes via the patch pipettes, which can also be referred to as aperture or aperture region in the sense of the invention, a sealing or sealing equipment, for example in the sense of a Gigaseal reached, then to perform so-called patch-clamp investigations , It is also conceivable to reverse the arrangement of the structure, and to lower the chip and the analysis hardware to the adherent cell culture on the second carrier.

Common to both variants is that the cell culture and analysis chip, i. that is, the second carrier and the first carrier can be spatially separated from one another until shortly before the actual measurement or patch clamp analysis. This may also serve, e.g. the cells during cultivation and shortly after to keep under optimal conditions, whereas the actual measurement process then optionally under physiological non-optimal conditions and thus takes place shortly before the measurement.

Some of the advantages of the new design according to the present invention can be described as follows: This results in a freely definable lowering or approach between stamp, so the second carrier, and the chip, so the first carrier. This allows an adjustable perfusion volume, and in particular its minimization, and also allows an adjustable perfusion speed between punch, ie second carrier, and chip, ie first carrier.

The perfusion can be supported integrated in or on the stamp or chip in the form of microfluidic channels and / or webs. By means of the possibly very small distances between punch and chip, for example in the range of 5 to 25 μπι, are very small volumes and very fast exchange rates of measuring solutions or perfusion solutions possible. Thus, a quick replacement of the active ingredients contained therein is conceivable. Construction, materials and processing of the chip or first carrier and / or the second carrier allow the formation of very high sealing resistances or seals, in particular in the so-called giga ohm range.

By structuring the second support, perfusions of individual stripe sections, e.g. in the sense of pipette strips, for adherent cells possible. It can be formed so-called positioning webs on the second carrier or stamp and / or the first carrier or chip, which allow self-alignment and / or self-organization in the alignment between the first and second carrier. The inventive technique also allows the integration of so-called contact pressure sensors to automate the lowering of the stamp and thus to make customer-friendly. In this case, elements of the piezoelectric technology can be used, it can, for. Piezo elements may be designed as pressure sensors integrated in the surface of the first carrier and / or the second carrier, if appropriate with a corresponding electrical connection to an intended control electronics.

The structures on the chip or first carrier and / or the stamp or second carrier, in particular in the respective surface, may be formed in channel and / or web shape. Thus, a targeted supply of perfusion solution or active solution to the biological objects to be examined, and in particular to be measured cells possible. The appropriate solution exchange can optionally be done very quickly. The movement of punches, ie the second carrier, and / or chip, ie the first carrier, in the x, y and z directions and by lateral torsional motion is possible by means of computer-aided algorithmics, the calculation of an optimal punch-to-chip alignment to bring a maximum number of adherent cells in contact with the on-chip patch pipettes, so the apertures.

Overall, there is an increase in throughput in the analysis of adherent cells. Furthermore, the separation of the chip, ie the first carrier and culture, ie the second carrier, makes sense, because it avoids the risk of chip failure in poor cultures. Furthermore, the separation of the chip, ie the first carrier and culture, ie the second carrier, allows the measurement of the positioning of cells chemically or structurally bound to the platform directly above the respective patch pipettes or apertures.

The sequence of FIGS. 1A to 1C shows, in a schematic and sectional side view, an embodiment of the electrophysiological measuring arrangement 100 according to the invention in various states which are adopted when using the measuring arrangement 100. It should be noted that the representation chosen there may not be true to scale.

It can be seen from FIG. 1A that by means of an intended actuator 80 along the spatial direction according to the arrow Z, a first carrier 12-1 having a lower side 12-1b and an upper side 12-1a, which is formed from a material 12-1 ' , relative to a second carrier 12-2 with a bottom 12-2a and a top 12-2b and a material 12-2 'can be moved, so that a defined approach between the first and second carrier 12- 1 and 12, respectively -2 can be performed in a controlled manner. By means of such a controlled approach, the biological object O to be measured, which is attached to the underside 12-2a of the second carrier 12-2, becomes the aperture area 10 and the aperture 14 of the first carrier 12-1 of the measuring arrangement 100 provided therein controlled way closer.

The structure of the biological object O to be examined, which is shown in FIGS. 1A-1C, is only representative in the sense of an individual cell. Representative for it also an ensemble of biological objects in the sense of a cell culture, a cell network or a cell lawn is meant.

In the state of FIG. 1A, the biological object O to be examined and the second support 12-2 are still relatively far away from the aperture region 10 and the aperture 14 and thus from the first support 12-1. In the intermediate state shown in FIG. 1B by the actuation of the actuator 80, an approximation of the second carrier 12-2 with the biological object to be examined O to the first carrier 12-1 and thus to the aperture area 10 and the aperture 14 takes place, so that now the wall area or the aperture wall 1 1 of the Aperture 14 has come into contact with its upper edge 11 lo with the membrane M of the biological object O to be examined.

Of course, the approximation of the first carrier 12-1 can also take place when the second carrier 12-2 is stationary. Also, both carriers 12-1 and 12-2 can be moved together toward each other. Furthermore, a reorientation is also possible, so that the second carrier 12-2 lies at the bottom with the biological objects O in FIGS. 1A to 1C, but then with its side 12-2a as the upper side, and so that then the first carrier 12 - 1 is oriented upwards, but then with the aperture area 10 and its side 12- la oriented downwards, namely the side 12-2a of the second carrier 12-2 and the biological object to be examined O associated and facing.

In the transition to the state according to FIG. 1C, the cell-attached mode of FIG. 1B is then changed to the whole-cell mode, namely by opening the membrane M of the biological object O to be examined, e.g. by a pressure surge with negative pressure across the aperture 1 with attached object O, e.g. in the state according to FIG. IB. Starting from the state shown in FIG. 1C, the arrangement of electrode 50 and counterelectrode 30 via the electrolyte bath 40 in the intermediate space between the first and second supports 12-1 and 12-2, the electrolytic bath 60 on the rear side 12-1b of the first Carrier, in which the counter electrode 30 is arranged, and the electrolyte ribbon 70 in the lumen of the aperture 14 carried out the electrophysiological measurement on the object to be examined O, as is also further illustrated in connection with FIGS. 12 and 13 below.

FIG. 2A shows in plan view an embodiment of a first carrier 12-1 and its upper side 12-la, in which the aperture region 10 has a plurality of apertures 14, which are in the manner of a grid or a matrix represented by the dashed lines Grid points thereof are arranged. Basically, other topologies are conceivable

FIG. 2B shows a view of an embodiment of the second carrier 12-2 and its underside 12-2a, which has a plurality of biological objects O to be examined, which likewise are in the manner of a matrix or a grid, which are again indicated by dashed lines, lie at corresponding grid points thereof, wherein the grid points at least approximately correspond to the grid points of the matrix arrangement for the apertures 14 of the first carrier 12-1 to speak so that each aperture 14 of the first carrier 12-1 is directly associated with a biological object O to be examined on the lower surface 12-2a of the second carrier 12-2 and when approaching the lower surface 12-2a of the second carrier 12-2 relative to the upper side 12-la of the first carrier 12- 1 at each aperture 14 with respect to the respective associated biological object a situation arises, as shown in connection with the sequence of Figs. 1A to IC for a single object O.

Finally, FIG. 2C shows a superimposition of the arrangements from FIGS. 2A and 2B, wherein in each case one of the apertures 14 is assigned a biological object O, so that when the first and second supports 12- 1 and 12-2 approach each other FIGS. 1A to 1C form a so-called multiple attachment and a plurality of or all individuals of the biological objects O to be examined can be examined electrophysiologically together or sequentially or also in interaction with one another in a measurement setup.

FIG. 3A shows a schematic and sectional side view of another embodiment of the electrophysiological measuring arrangement 100 according to the invention.

A basic element of this embodiment is the first carrier 12-1, which may also be referred to as the first base 12-1 or as the first substrate 12-2. This first carrier 12-1 divides an electrolytic bath 40, 60, 70 provided during the measurement into at least two compartments, the first compartment 60 facing the underside 12-1b of the first carrier 12-1 or substrate 12-1 and the second compartment 40 faces the top 12-1 of the first carrier 12-1 or substrate 12-1.

In the first carrier 12- 1, the so-called aperture area 10 is incorporated. This has at least one aperture 14, namely e.g. in the manner of a through-hole, which completely penetrates the first carrier 12-1 locally in its thickness or layer thickness direction, namely in the direction from the upper side 12-la to the lower side 12-lb. In the region of the aperture 14, a portion 70 of the electrolyte bath 40, 60, 70 is also provided.

Overall, therefore, there is a fluid-mechanical connection between the upper side 12-la and the lower side 12-lb via the aperture 14 of the aperture region 10 and correspondingly via the optionally provided conductivity of the Elektrolytbads, it is often in the application of a physiological solution, including an electrical connection.

In the embodiment according to FIG. 3A, a counterelectrode 50 connected to a line 51 is arranged in the upper-side compartment 40 to a measuring electrode 30 provided on the lower side 12-1b of the first carrier 12-1. The measuring electrode 30 is thus located in the lower compartment 60 of the electrolyte bath 40, 60, 70 and is connected by a line 31. The lines 51 and 31 to the counter electrode 50 and the measuring electrode 30 are isolated and lead to a corresponding control and measuring circuit, which is not shown here.

The first substrate 12-1 or the first carrier 12-1 are formed by a first carrier material 12-1 '.

The wall region 11 as such forms in the embodiment of FIGS. 1A and 1B a closed wall running in the manner of an inner lateral surface with an inner side 10i, 11c or inner wall 10i, 11e and an outer side 10a, 11a or outer wall 10a, l Ia. In this way, an aperture 14 of the aperture region 10 of the electrophysiological measuring arrangement 100 according to the invention is formed, the inner side or the inner wall 10 '. On the other hand, the outer wall or outer side 10a, 11a of the wall region 11 faces away from the aperture 14, but faces the compartment 40 of the electrolyte bath 40, 60, 70.

In the embodiment of FIG. 1A, the wall region 11 rises exclusively above the upper side 12-1a of the first carrier or substrate 12-1. On the underside 12-1b of the first substrate or carrier 12-1, the aperture region 10 is formed quasi-planar , However, such a structure is not mandatory, and Figs. 4 to 8 show modified embodiments thereof, which will be described later in detail.

As mentioned above, the wall portion 1 1, which forms the aperture 14, formed in the manner of a lateral surface of a geometric body. According to FIG. 3B, in conjunction with FIG. 3A, this lateral surface can originate from a vertical circular cylinder as a basic shape.

The training as a lateral surface of a vertical circular cylinder as a basic form is not mandatory. FIGS. 3C and 3D show, instead of a cylinder form a schematic plan view as the base square so that spatially results in a vertical square prism or a prism with a base in the manner of an oval, a quasi square or rectangle with rounded corners, as shown in Fig. 3D.

There are basically any base shapes imaginable. However, owing to the circumstances described above, in which surface roughness and sharp edges should be avoided, straight shapes with rounded structures are available, for example the embodiment according to FIG. 3B, which has a vertical circular cylinder as the geometric basic shape.

FIGS. 4 and 5 show, analogously to FIG. 3A and likewise in schematic and sectional side view, other embodiments of the measuring arrangement according to the invention, in which there is a difference in that, as shown in FIG. 4, the wall region 1 1 for the aperture 14 is flush with the Top 12- la of the first substrate 12-1 closes and protrudes exclusively on the bottom 12- lb of the first substrate 12-1 out, so that a total of a kind of invagination from the top 12- la inwardly to the compartment 60 for the aperture 14 is formed ,

5, a portion of the wall portion 1 1 of the aperture 14 rises from the top 12- la of the first substrate 12- 1 in the compartment 40, on the other hand, however, a part of the wall portion 1 1 of the bottom 12- lb of first substrate 12-1 into the compartment 60.

The heights above the upper side 12-1a and above the lower side 12-1b, by which the wall area 11 for the aperture 14 each rise, may be identical. This is not mandatory. In Fig. 5, they are designed differently. In the embodiments of FIGS. 3 and 4 and 5, the cross section or diameter along the extension direction of the wall portion 1 1 is perpendicular to the top 12- la or perpendicular to the bottom 12- lb of the first substrate 12- 1 constant in its course. Alternatively, tapering or widening cross-sectional profiles can be provided. This is shown in the sequence of FIGS. 6 to 8, wherein FIG. 6 shows an embodiment of the measuring arrangement according to the invention, which corresponds to the embodiments of FIGS. 1 a to C and 3 A, but with a cross-section. For the aperture 14, which narrows with increasing distance from the upper side 12-la of the first carrier 12-1.

By contrast, in the embodiment of FIG. 7, the aperture 14 is designed analogously to this and, in comparison to the embodiments of FIGS. 1A to C and 3A, such that the cross-sectional profile tapers at a distance from the underside 12 - 1b of the first substrate 12.

In a combination of the embodiments of FIGS. 6 and 7 and in an analogous view to the embodiment of FIG. 4, FIG. 8 shows an aperture 14 in which the diameter of the aperture 14 is at the height of the first substrate 12-1 and tapers with increasing distance both from the upper side 12-la of the first substrate 12-1 and from the lower side 12-1b of the first substrate 12-1.

According to the embodiment of FIGS. 1A to C and 3A, the measuring electrode 30 is strongly adjacent to the lead 31 and partially inserted into the aperture region 10 having the aperture 14. The position of the measuring electrode 30 can be varied, for example, it can be more guided into the electrolyte region 70 in the aperture 14 or be removed further therefrom.

FIGS. 9A to 9E show a schematic and sectional side view of various measuring principles that may be used in the electrophysiological measuring arrangement 100 according to the invention and the corresponding electrophysiological measuring method according to the invention.

Starting from the situation shown in Fig. 9A, in which by a slight suction of the electrolyte bath 40, 60, 70 through the measuring aperture 14, ie with a suction from the compartment 40 via the compartment 70 in the aperture 14 to the compartment 60 out, is a biological object O, in this case, for example, a cell sucked and approximated to the aperture 14.

The mechanism of approach may also be done in another manipulative manner, for example by means of a separate pipette, laser tweezers or the like.

By means of a slight negative pressure, according to FIG. 9B, the cell is deposited without any destruction and completely on the measuring aperture 14. Will in this state a measurement is performed, this is called the cell-attached mode.

By means of a mechanical pull, starting from the situation described in FIG. 9B, a membrane spot can be torn out of the membrane of the biological object O, so that according to FIG. 9C the torn-out membrane patch, a so-called patch, in the sealed or seamed state in FIG the measuring aperture 14 remains and actually forms the measuring object O. As a result of the tearing out, the side of the membrane spot previously arranged in the interior points outwards, ie towards the compartment 40. For this reason, this measurement mode is also called Inside-Out mode.

On the other hand, starting from the situation shown in Fig. 9B, it can be replaced by a new negative pressure, e.g. the cell O are opened in the manner of a pressure surge so that a transition takes place from the so-called cell-attached mode to the whole-cell mode according to FIG. 9D, in which the measuring electrode 30 has direct access to the entire cell interior. That is, the cell interior is open to compartments 70 and 60, but substantially isolated from compartment 40.

From the situation according to FIG. 9D, namely the whole-cell mode, it can be achieved by mechanical pulling that in turn a membrane fragment is torn out of the membrane of the biological object. Since the whole-cell mode was present before, there is a certain probability that the torn-out membrane spot remains in the measuring aperture 14 in such a way that it can serve as the actual measuring object O and thereby according to FIG. 9E the outside of the cell membrane or the membrane of the membrane biological object also remains outside. In this case, one speaks of the outside-out mode of the measuring arrangement 100.

10 shows in detail the geometrical situation which is present in the case of a sealed or sealing attachment of a biological object O to be measured between its membrane M and the measuring aperture 14 and in particular the inner wall 10i, 11b of the wall area 11.

The mode of FIG. 10 is again a whole-cell mode in which the entire cell O is opened with its interior towards the measuring electrode 30 and towards the electrolyte compartments 60 and 70. This embodiment of the The electrophysiolgic measuring arrangement 100 according to the invention is designed here in the manner of a patch pipette.

According to the invention, the sealing resistance between the cell membrane M of the biological object O to be measured and the inner wall 10i, 11b of the wall area 11 of the measuring aperture 14 is improved in that after controlled approach of the biological object O by means of the second carrier 12-2 via the contact tuator 80 the interaction with the outside of the membrane M of the biological object O is promoted.

In the embodiment according to FIG. 10, the first substrate or the first carrier 12-1 mediated by the second substrate or the second carrier 12-2 is quasi occupied by a layer of cells O '. By means of the interaction with the aperture wall 11 according to the invention, a single cell O of the ensemble of cells O 'as a measurement object in whole-cell mode is accessible analogously to the situation according to FIG. 9D with improved sealing or sealing attachment of an electrophysiological measurement ,

11 shows an arrangement of the measuring arrangement 100 according to the invention, in which the wall region 1 1 forming an aperture 14 is formed by an edge region, so that the aperture 14 acts as a planar hole in the underlying first substrate or substrate 12. 1 is designed to promote the attachment and increase the sealing resistance.

FIG. 12 shows in the upper and lower regions respectively a graph with a plurality of measurement curves which were derived with an electrophysiological measurement arrangement according to the present invention, wherein the biological objects to be examined are neurons, ie nerve cells, and wherein the measurement curves demonstrate the so-called evoked potentials in their time course. In this case, a voltage pulse is applied via the arrangement of the electrode 50 and counterelectrode 30 when the neuron is attached to the measuring aperture 14 as a biological object O initially and then the cell response is measured in terms of a derived potential over its time course. The two graphs of FIG. 12 superimpose a large number of such evoked potentials at different initial voltage pulses under different conditions. FIG. 13 demonstrates a so-called single-channel measurement on a single cell using the electrophysiological measurement arrangement according to the present invention. In this case, a single cell is supplied as a biological object to be examined O a single aperture 14, so that there is a sealing attachment, ie a gigaseal. Thus, there arises a situation as shown in the sequence of Fig. 1A to IC. Then, under physiological or other conditions, the time profile of the electrical potential difference across the membrane M is measured. There are so-called channel-forming proteins in the cell membrane. Roughly speaking, these have the property of being in an open or closed state. In the open state, ions, optionally selectively, may flow through a pore. When closed, this is not possible, the pore is then closed.

Under constant conditions, in the open state, a very specific ion current flows across the membrane, which can be measured as an electric current of a certain height and direction. This must be the same or approximately the same for channel-forming proteins and constant conditions.

Fig. 13 now demonstrates a so-called single-channel measurement on a cell, wherein the solid line "0" represents the so-called zero current line and represents the state in which there is no single channel in the cell membrane in the open state, so net no ion current across the membrane M is clearly visible, that - starting from the zero line "0" - there are various intermediate states in which there are certain distances to the zero line in the course of the electric current. The dashed line "1" indicates states in which a single channel is opened, whereas the dashed line "2" indicates states in which two channels are simultaneously opened. LIST OF REFERENCE NUMBERS

10 aperture area

10 a outer wall, outer wall area, outside of the aperture area 10 10 inner wall, inner wall area, inside of the aperture area 10

1 1 wall area, wall, aperture wall

I Ia outer wall, outer wall area, outside of the wall area 1 1 1 Ii inner wall, Aperturinnenwand, inner wall area, inside the

Wall area 1 1

l lo upper edge / top edge outer wall, outer wall area, outside of

Aperture wall 1 1

l lu lower edge / lower edge outer wall, outer wall area, outside of the aperture wall 11

12- 1 first carrier, first base, first base substrate, first substrate

12-1 'carrier material, substrate material of first carrier 12-1

12- la top, front of the first carrier 12- 1

12- lb bottom, back of first support 12- 1

12-2 second carrier, second base, second base substrate, second substrate 12-2 'carrier material, substrate material second carrier 12-2

12-2a top, front of second carrier 12-2

12-2b base, back side 12-2 second

14 aperture, measuring aperture

20 area with modified, in particular increased concentration; Concentration-modified area

30 measuring electrode, measuring electrode arrangement

31 measuring line, line

40 electrolyte compartment, electrolyte bath

50 counter electrode, counter electrode arrangement

51 line

40 Elektrolytkompartiment, electrolyte bath to the top 12 la of the first

Carrier 12-1

60 electrolyte compartment, electrolyte bath to the rear 12- lb of the first

Carrier 12-1

70 electrolyte compartment, electrolyte bath in the lumen of the aperture 14

80 actuator

100 electrophysiological measuring arrangement

M membrane of the biological object O

O biological object, cell, liposome, vesicle, micelle, oocyte, target O 'biological object, cell, liposome, vesicle, micelle, oocyte x spatial direction

X rotation axis

y spatial direction

Y rotation axis

z spatial direction

Z rotation axis

Claims

claims

1. electrophysiological measuring arrangement (100),

(a) with a first support (12-1) having an aperture region (10) which is formed in the region of the first support (12-1) for the controlled sealing attachment of a biological object (O) and which is provided with at least one aperture ( 14) and with a wall portion (1 1) is formed, which surrounds the aperture (14), and

(b) with a second carrier (12-2) which is formed at least on a side (12-2a) associated with the first carrier (12-1) for the adherence and / or culturing of biological objects (O) to be examined,

(c) wherein the first and the second carrier (12- 1, 12-2) - in particular controllable - with the said first carrier (12- 1) associated side (12-2a) of the second carrier (12-2) the first Carrier (12- 1) and the aperture region (10) of the first carrier (12- 1) are formed substantially facing one another at an approachable.

2. Electrophysiological measuring arrangement (100) according to claim 1,

which for the approach of the first carrier (12-1) and / or the second carrier (12-2) with respect to each other - in particular in a controlled manner - at least one actuator (80) through which the first carrier (12-1 ) and / or the second carrier (12-2) are movable relative to each other, and / or means for self-organization, self-leveling, and / or self-adjustment in the approach and / or alignment, in particular positioning marks or webs, contact or pressure sensors.

3. electrophysiological measuring arrangement (100) according to claim 2,

in which the respective actuator (80) results in a linear displacement of the first carrier (12-1) and / or the second carrier (12-2) along one or more of the three spatial directions (x, y, z) and / or for rotation of the first carrier (12-1) and / or of the second carrier (12-2) is formed around one or more axes of rotation (X, Y, Z) corresponding to the three spatial directions (x, y, z).

4. Electrophysiological measuring arrangement (100) according to one of the preceding claims,

in which at least the side (12-2a) of the second carrier (12-2) associated with the first carrier (12-1) is formed with or made of a soft and / or flexible material, in particular with or from PDMS or the like.

5. Electrophysiological measuring arrangement (100) according to one of the preceding claims,

in which the aperture region (10) is formed in the region of the first carrier (12-1), which has an upper side (12-1a) and a lower side (12-1b), and

in which a respective wall region (1 1) forming an aperture (14) protrudes partially or completely with respect to the upper side (12-la) and / or with respect to the lower side (121 -b) of the first carrier (12-1) ,

6. Electrophysiological measuring arrangement (100) according to one of the preceding claims,

in which a wall region (11) forming an aperture (14) is designed in the manner of a lateral surface or as a combination of lateral surfaces, in particular based on a jacket of a cylinder, a prism, a truncated cone and / or a truncated pyramid and / or

in which a wall region (11) forming an aperture (14) is formed with or from a material from the group of materials comprising glass, quartz glass, silicon, carbon and combinations thereof.

7. Electrophysiological measuring arrangement (100) according to one of the preceding claims,

in which the diameter of the aperture (14) and in particular the inner diameter of the aperture (14) forming wall portion (1 1) has a value in the range of about 1 μπι to about 50 μπι and / or

at which of the one aperture (14) forming wall portion (1 1) above the top (12- la) or the bottom (12- lb) of the first carrier (12- 1) a height or depth in the range of about 0 μπι to about 20 μηα has.

8. Electrophysiological measuring arrangement (100) according to one of the preceding claims,

in which a measuring electrode (30) in the region (70) or within the aperture (14) or in a region (60) on the underside (12- lb) of the first carrier (12- 1) is provided and

in which a counter-electrode (50) is provided outside the aperture (14) and in the region (40) on the upper side (12-la) of the first carrier (12-1).

9. Electrophysiological measuring arrangement (100) according to one of the preceding claims, in which the aperture region (10) of the first carrier (12-1) has a plurality of apertures (14), in particular in a grid or matrix-like arrangement and / or analogous organotypic forms such as the hippocampus or other biological Structures, and

wherein the second carrier (12-2) on the side (12-2a) associated with the first carrier (12-1) has an arrangement of positioning recesses and / or projections corresponding to the arrangement of the plurality of apertures (14) and / or or fixation of a biological object (O),

wherein, in particular, directly adjacent positioning recesses and / or projections are respectively connected by a channel and / or web in the side (12-2a) associated with the first carrier (12-1).

10. An electrophysiological measuring method using an electrophysiological measuring arrangement (100) according to one of claims 1 to 9,

which has the steps:

(1) providing the first carrier (12-1) and the second carrier (12-2) in a fluid measuring medium (40),

(2) providing the second support (12-2) with adhered or suspended biological objects (O) on the second support (12-2), optionally by culturing the biological objects (O) on the second support (12-1),

(3) positioning and aligning the first and second supports (12-1, 12-2) with each other, in particular in a controlled manner, and thereby - in particular controlled - approaching the first and second supports (12-1, 12-2) in such a way that at least one biological object (O) is thereby approximated to the region of at least one aperture (14) so that contact between the membrane (M) of the biological object (O) and the aperture (14) takes place at least directly can or takes place

(4) suction and suction of the membrane (M) located in the region of the aperture (14) biological object (O) by means of negative pressure on the aperture (14), (5) characterized attraction of the sucked and sucked portions of the membrane

(M) and sealingly attaching the sucked and sucked portions of the membrane (M) on the inner wall (I ii) of the aperture (14) and

(6) Performing an electrophysiological measurement on the biological object (O) located in the region of the aperture (14).

1 1. A method according to claim 10,

in which a randomly distributed cell ensemble or network on the second carrier (12-2) by means of the controlled positioning and alignment of the First and second carrier (12-1, 12-2) is examined by means of an electrophysiological measurement.

12. The method according to any one of claims 10 or 1 1,

in which, by approaching the first and second supports (12-1, 12-

2) with respect to each other in a controlled manner by means of the controlled positioning and alignment of the first and second supports (12-1, 12-2) the total volume of a free measuring space is reduced, in particular to thereby define a measuring environment of the biological object (O ) to be rapidly changed, in particular by rapid perfusion and / or concentration jump experiments, and / or to rapidly change the experimental conditions for the biological objects (O) to be examined.

13. The method according to any one of claims 10 to 12,

in which a tilting of the first and / or the second carrier (12-

1, 12-2) via a self-leveling of the respective carrier (12-1, 12-2) takes place, in particular when the respective carrier (12-1, 12-2) consists of a soft and / or flexible material, in particular (A) by means of a joint between the second carrier (12-2) and an actuator (80), (b) by a flexible material, such as PDM or others, and / or (c) small and spaced defining and holding fufions between the first and second supports (12-1, 12-2).