EP2089508A1 - Dispositifs et procédés pour analyses électrophysiologiques de cellules - Google Patents

Dispositifs et procédés pour analyses électrophysiologiques de cellules

Info

Publication number
EP2089508A1
EP2089508A1 EP07819499A EP07819499A EP2089508A1 EP 2089508 A1 EP2089508 A1 EP 2089508A1 EP 07819499 A EP07819499 A EP 07819499A EP 07819499 A EP07819499 A EP 07819499A EP 2089508 A1 EP2089508 A1 EP 2089508A1
Authority
EP
European Patent Office
Prior art keywords
capillary
holding element
rod
liquid
patch
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP07819499A
Other languages
German (de)
English (en)
Inventor
Steffen Hering
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Universitaet Wien
Original Assignee
Universitaet Wien
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Universitaet Wien filed Critical Universitaet Wien
Publication of EP2089508A1 publication Critical patent/EP2089508A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/48707—Physical analysis of biological material of liquid biological material by electrical means
    • G01N33/48728—Investigating individual cells, e.g. by patch clamp, voltage clamp

Definitions

  • the invention relates to a liquid holder device for electrophysiological examinations of biological cells, in particular a liquid holder device having the features of the preamble of claim 1.
  • the invention further relates to an electrophysiological measuring device which is equipped with such a liquid holder device and in particular comprises a patch-clamp pipette.
  • the invention also relates to a method for the electrophysiological examination of a biological cell using such a liquid holder device.
  • the patch-clamp device contains a capillary-shaped pipette (patch-clamp pipette), at the tip of which a cell can be aspirated and measured.
  • the patch-clamp pipette contains a measuring electrode for deriving an electrophysiological potential from the cell or for measuring transmembrane ion currents.
  • the ion channels are important targets for drugs.
  • a variety of drugs act selectively on certain ion channels. so that a tool for pharmacological examinations is available with the patch-clamp technique.
  • Drop holder is surrounded.
  • the desired liquid environment of the capillary tip can be formed with a drop of liquid trapped by capillary forces in the drop holder.
  • a disadvantage of the conventional drop holder according to US 2005/0241940 A1 consists in the coaxial relative movement of pipette tip and drop holder, which have a small distance (capillary gap) in all relative positions. To avoid damage to the pipette or cell, therefore, a complex precision drive is required.
  • Another disadvantage is the obstruction of the perfusion of the cell in the altered drug solution, which is initially shielded by the drop holder on all sides of the cell becomes. This results in a relatively high expenditure of time for the perfusion, which is disadvantageous for the reproducibility and comparability of a plurality of patch-clamp measurements on a cell.
  • a further object of the invention is to provide an improved measuring device, in particular for patch-clamp examinations.
  • a generic liquid holder means for forming a liquid environment on a capillary tip of a patch-clamp pipette is arranged to provide a holding element for receiving a liquid drop, which is arranged separable from the capillary when used for liquid holding with a patch-clamp pipette.
  • the at least one rod for holding the retaining element is formed so that the retaining element is separable in particular from the capillary.
  • the holding element can be positioned with the rod on or next to the capillary tip (conditioning position) and can be separated from the capillary (release position) so that the capillary tip is free and a distance between the holding element and the capillary tip is greater the width of a capillary gap.
  • the retaining element In contrast to the conventional drop holder, the retaining element
  • a number of advantages can be achieved both in terms of the requirements and accuracy of movement of the retainer and with respect to the intended use of the patch-clamp pipette.
  • the rod to which the holding element is attached is adapted to be pivotably fixed to a capillary holder of the patch-clamp pipette.
  • the rod preferably has a pivot joint on an end opposite the retaining element.
  • the retaining element has a free surface on at least one side, which can be approximated laterally to the capillary tip, ie radially with respect to a longitudinal direction of the capillary.
  • the rod when the liquid holder device according to the invention is attached to a patch-clamp pipette, the rod is pivotable relative to the longitudinal direction of the capillary of the patch-clamp pipette.
  • the holding element can be pivoted by pivoting of the rod from an applied state (conditioning position), in which the holding element is separated from the capillary tip by a capillary gap or abutting the capillary tip, by a radial movement in a protruding state (release position), in which the holding element, the Kapil- larspitze, z. B. for further observations, measurements or
  • the axial movement of the conventional drop holder is replaced by a radial movement of the holding element according to the invention.
  • the retaining element for holding the liquid droplet for the formation of the liquid environment at the capillary tip is arranged under the action of the gravitational force, wherein the retaining element is releasably secured to the rod.
  • the holding element forms a support on which a quantity of liquid is carried.
  • larger amounts of liquid can be provided to condition the capillary tip with improved stability and reliability.
  • the holding element may have a unilaterally open, planar or curved shape. This results in a further important advantage improved perfusion in the transfer of a liquid-enclosed cell at the top of the capillary in a changed environment medium.
  • the cell is only partially shielded by the retaining element from the new surrounding medium, so that the droplet on the droplet holder dilutes rapidly due to liquid flow and diffusion.
  • the effect of the substance in the new surrounding medium e.g. B. a new drug solution to be examined after a time that is shorter compared to the time required for perfusion with a conventional drop holder.
  • the holding element may be a planar or curved component which, in the applied state of the liquid-holding device, is arranged adjacent to the capillary tip or partially surrounds it in an azimuthal manner.
  • the retaining element is sheet-shaped, z. B. formed in the form of a glass or plastic lamella.
  • the holding element is set up to receive (hold) a drop of liquid.
  • the liquid drop is stably captured in the first embodiment of the invention under the action of capillary forces. It can be provided that the drop is held by the capillary forces exerted in the applied state of the liquid holder device from the tip of the capillary and the holding element together capillary forces.
  • an embodiment of the Invention in which the holding element is adapted to hold the liquid drop alone without further solid interfaces under the action of capillary forces. In this case, even in the protruding state of the liquid holder device, a drop of liquid can be held securely on the holding element and thus avoided that gas bubbles settle on the holding element.
  • the holding element comprises a sheet-like component, advantageously a relatively large amount of liquid in the vicinity of the tip of the capillary can be obtained. This reduces the risk of inadvertent drying out of the cell during transfer into a changed surrounding medium.
  • the holding element is formed by a portion of a hollow line cut in the axial direction.
  • the wall of the hollow conduit forms the retaining element open on one side.
  • the hollow cylinder can, for. B. have a circular or elliptical base.
  • the drop holder is designed to surround the pipette tip in the applied state of the liquid holder device so that it is arranged in the center or focal point of the circular or elliptical base surface.
  • the profiled plate has z. B. a shape with several flat faces, which are angled towards the top of the capillary, or a profile, which is adapted to the shape of the capillary tip of the capillary.
  • the structure of the liquid holding means can be simplified.
  • the electrode can be used as a reference electrode for patch-clamp measurements.
  • the electrode on the holding element or a line connected to the electrode is preferably connected to the rod of the drop holder.
  • the rod with the holding element and the electrode form an integrated component.
  • the liquid holder device according to the invention can be easily used in automated processes.
  • adjusting movements for transferring the drop holder from the applied to the protruding state can be carried out without difficulty with a setting device which is connected to a patch clamp.
  • the liquid holder device is equipped with an independent drive device.
  • the drive device is z. B. for piezoelectric pivoting of the rod of the liquid holder device and preferably provided on the pivot joint to achieve a large travel.
  • an important advantage of the invention is the compatibility of the liquid holder device according to the invention with different capillary and in particular pipette forms.
  • the swivel joint of the liquid holder device is arranged for releasable connection to the capillary. tet.
  • existing patch-clamp pipettes can be retrofitted with the liquid holder device.
  • the liquid holder means for releasable connection with the capillary, z. B. the patch-clamp pipette is set up. Alternatively, a fixed connection may be provided.
  • the rod of the liquid holder means is preferably adapted for rigid attachment to the capillary or the capillary holder of the patch-clamp pipette, so that advantageously simplifies the structure of the liquid holder device and the patch-clamp pipette.
  • the retaining element has the shape of a cup, in the wall of which an insertion opening for receiving one end of the rod is provided. If the insertion opening and the interior of the cup are connected via a contact channel according to a further preferred variant of the second embodiment of the invention, there are advantages for the measurement with a reference electrode formed by the rod or connected to the rod.
  • the holding element of the liquid-holding device according to the invention is in accordance with the preferred applications of the invention in cell biological studies for receiving a small amount of liquid (volume in particular 0.05-
  • aqueous salt solution such as a physiological solution, an aqueous solution or suspension with biological macromolecules or with cells or a aqueous-oily emulsion.
  • the catching action of the holding element can be advantageously improved in both embodiments of the invention even if the holding element carries a hydrophilic or an oleophilic inner coating.
  • the inner coating is formed by hydrophilic or oleophilic substances, as z. B. of functionalized substrates in cell biology are known.
  • An electrophysiological measuring device which has a capillary with a capillary tip for receiving a biological cell and a liquid holder device according to the invention connected to the capillary constitutes an independent subject of the invention.
  • the capillary is preferably part of a patch-clamp pipette or a microelectrode device ,
  • the patch-clamp pipette preferably has a capillary holder on which the capillary can be detachably fixed.
  • the capillary as a gripper for further manipulations, for. B. be used in the manufacture of the capillary.
  • the capillary is connected via a conical plug connection with the capillary holder.
  • the meter is equipped with a drive mechanism designed to move and position the patch-clamp pipette and fluid holder, it can provide advantages for automated operation of the meter.
  • the measuring device can be equipped with a pipette pulling device, which is set up to produce the capillary.
  • the capillary can thus be used immediately before an electrophysiological measurement. be prepared and used free from damage or contamination.
  • the above-mentioned object is achieved by the general technical teaching to carry out a method for the electrophysiological examination of a biological cell using the liquid holder device according to the invention.
  • patch-clamp measurements can be performed during the transfer of a cell to and in a changed environment medium during perfusion.
  • Figures 1 to 4 variants of the first embodiment of the liquid holder device according to the invention
  • Figures 5 and 6 a schematic illustration of the transfer of a cell according to the invention into a changed surrounding medium using the liquid holder device according to the first embodiment of the invention
  • FIG. 7 shows a further variant of the first embodiment of the liquid holder device according to the invention.
  • FIG. 8 shows a preferred variant of the retaining element used in the second embodiment of the invention
  • Figure 9 variants of the second embodiment of the liquid holder device according to the invention
  • FIG. 10 shows a schematic illustration of the transfer of a cell according to the invention into a changed surrounding medium using the liquid holder device according to the second embodiment of the invention
  • FIG. 11 illustrations of additional parts of a measuring device for electrophysiological examinations
  • FIG. 12 shows a schematic illustration of a capillary holder of a preferably used patch-clamp pipette
  • FIG. 13 Illustrations of the filling of the capillary of a patch-clamp pipette with a filling device
  • FIG. 14 shows a schematic illustration of a capillary holder of a patch-clamp pipette.
  • the invention will be described below by way of example with reference to a preferred embodiment of the liquid holder device attached to a pipette tip of a patch-clamp device. Details of the patch-clamp technique are not described here as they are well known in the art (see, for example, OP Hamill et al., "Pflugers Arch.” Vol. 391 (2), 1981, p 85-100).
  • the liquid holder device can be used not only with a patch-clamp pipette, but also with other instruments for electrophysiological investigations combined, such.
  • FIG. 1A shows a liquid holder device 10 with the holding element 11.1, the rod 12.1 and the swivel joint 13 according to a variant of the first embodiment of the invention.
  • the liquid holder device 10 is shown in connection with a patch-clamp pipette 20 in the protruding state of the holding element 11.1.
  • the holding element 11.1 is separated in this state from the capillary tip of the patch-clamp pipette 20.
  • FIG. 1B shows an enlarged sectional view of the holding element 11.1, which is set up to receive a liquid drop (see FIG. 4).
  • the patch-clamp pipette 20 is constructed in a manner known per se with a capillary 21, which in particular has a free capillary tip 22 for aspirating a biological cell 2 and an integrated measuring electrode (not shown) and is fastened to a capillary holder 23.
  • the capillary tip 22 typically has a tip diameter in the ⁇ m range.
  • the capillary holder 23 is constructed, for example, as illustrated in FIG.
  • the composite of the liquid holder device 10 and the patch-clamp pipette 20 forms a measuring device 30 according to the invention (partially shown), which furthermore has line connections for connection to a control device and is optionally equipped with a drive device (see FIG. 7).
  • the holding element 11.1 has the shape of an axially truncated hollow cylinder with an elliptical base. With the holding element 11.1, a semi-tubular chamber (so-called half-tube chamber) is formed which is set up to receive the capillary 21 of the patch-clamp pipette 20 and a drop of liquid. On the inside of the holding element 11.1, the electrode 14 is arranged. That's on the outside Holding element 11.1 connected to the rod 12.1. The electrode 14 extends along the length of the rod 12.1.
  • the holding element 11.1 consists of glass or plastic (eg PVC) with a wall thickness of approx. 200 microns to 1 mm and a length in the longitudinal direction of the rod 12.1 of approx. 3 mm to 10 mm.
  • the lateral opening of the holding element 11.1 has a width of approx. 1.5 mm to 3 mm.
  • the electrode 14 is formed by a metal layer or a metal wire on the side (in particular inside) of the holding element 11.1, which faces the capillary 21.
  • the electrode 14 is z. B. from silver / silver chloride (Ag / AgCl) or from another electrically conductive material, for. As carbon or platinum.
  • the inside of the holding element 11.1 may be hydrophilic coated.
  • the electrode may consist of agar-agar and an Ag / AgCl electrode part, the agar-agar being connected to the electrode part via a salt bridge.
  • the electrode may be partially coated or disposed on the outside of the holding member 11.1, so that a diffusion path z.
  • B. Ag / AgCl is formed to the cell.
  • the rod 12.1 serves to hold the retaining element 11.1.
  • the rod 12.1 is a straight, elongate member having a longitudinal direction, from which the holding element 11.1 projects radially.
  • the holding element 11.1 opens in the radial direction relative to the longitudinal direction of the rod 12.1.
  • the rod 12.1 is connected to the pivot joint 13. tied, while the holding element 11.1 is attached to the second, free (distal) end of the rod 12.1.
  • the rod 12.1 consists of an inert, electrically insulating material, such. As plastic (eg., PE) or a ceramic.
  • the electrode 14 or a line connection connected to it is integrated in the bar 12. 1 or fastened on its surface.
  • the electrode 14 and the rod 12.1 are not two different components, but the rod forms the electrode. If the rod 12.1 and possibly the holding element 11.1 are adapted to form the electrode, these consist at least partially of a conductive material, such as. From Ag / AgCl. When using agar-agar, the electrode 14 (or according to the
  • Bar 12.1 preferably a glass tube in which the jelly-like agar-agar is arranged, which then passes into a salt bridge.
  • the pivot joint 13 includes z. As a hinge or a
  • Thin-film joint which is connected on the one hand to the rod 12.1 and on the other hand to the patch-clamp pipette 20 or a corresponding clamping device.
  • FIG. 1A schematically illustrates a piezoelectric drive device 15, which is set up for pivoting the rod 12. 1 about an axis perpendicular to the longitudinal direction of the patch-clamp pipette 20.
  • the drive device 15 is z. B. between a surface of the patch-clamp pipette 20 and the rod 12.1.
  • a piezoelectric element of the drive device 15 expands, so that the rod 12.1 can be pivoted.
  • FIG. 1C shows a further variant of a holding element 11.1 with a curved shape, wherein the holding element 11.1 is provided for detachable connection to the bar 12 of the liquid holder device 10 (see FIG. 1A).
  • the holding element 11.1 z. B. formed by injection molding with an insertion 11.11 and a semi-tubular chamber for receiving the capillary.
  • the rod 12, which can simultaneously form the reference electrode, is inserted into the insertion opening 11.11.
  • the electrical contact with the chamber, which is filled with liquid in the state of approaching the capillary tip, is produced by a contact groove 11.12.
  • the variant of the holding element 11.1 shown in FIG. 1C can be modified so that the chamber is tubular and can completely surround the capillary tip (not shown).
  • the holding element 11.1 has the shape of a cup with an open bottom. Because of the detachable connection with the rod 12, the holding element 11.1 can be separated from the capillary tip.
  • the holding element 11.1 is dimensioned so that the liquid is held in the holding element 11.1 by the action of capillary forces.
  • a holding element 11.1 in the form of a flat plate, z. B. be provided from glass or plastic.
  • the planar holding element 11.1 carries on the side facing the capillary, the electrode 14. With a suitable design of the rod 12.1, the holding element 11.1 may be integrally connected to the end of the rod 12.1.
  • the flat plate of the holding element 11.1 can be removed in the axial direction of the capillary 21. be angled ( Figure 3).
  • the holding element 11.1 adapted to the shape of the capillary tip 22 of the capillary 21.
  • the liquid droplet 1 on the holding element 11.1 is smaller than in the variant according to FIGS. 1 and 2, and the dead volume around the cell 2 is correspondingly reduced.
  • a more complex shape according to the outer contour of the capillary tip 22 may be provided.
  • FIG. 3 furthermore illustrates that the holding element 11.1 and with it the electrode can abut the capillary 21 directly.
  • the rod 12.1 is adjusted with the drive device 15 (FIG. 1A) such that the electrode is arranged in the immediate vicinity of the capillary tip 22.
  • FIGS. 4A and 4B show the first embodiment according to FIG. 1 in the applied state of the liquid holder device 10 with a liquid drop 1 and a cell 2. Between the capillary tip 22 and the inside of the holding element 11.1 there is only one capillary gap, or the inside of the holding element 11.1 touches the capillary tip 22. By the capillary forces, the liquid drop 1 is held on the capillary 21, even if the patch-clamp pipette 20 is withdrawn from a liquid surrounding medium.
  • FIGS. 5 and 6 the sequence of a method according to the invention for the electrophysiological examination of a biological cell using the first embodiment of the invention is illustrated schematically with further details.
  • Cells of a cell culture 3 are located on the bottom of a first culture vessel 40, which is filled with a first surrounding medium 4.
  • the surrounding medium 4 comprises z.
  • a cell 2 is removed in a conventional manner from the cell culture 3.
  • the cell removal is preferably carried out while the liquid holder device 10 is in the protruding state and the capillary 21 of the patch-clamp pipette 20 is moved freely to the cell culture 3, manipulated and can be observed with the image recording device 60 shown schematically.
  • the movement of the liquid holder device 10 and the patch-clamp pipette 20 takes place with a schematically shown drive device 50.
  • the drop 1 at the tip 22 of the patch-clamp pipette 20 is rapidly diluted by the second surrounding medium 5.
  • the measurement of cell potentials or ion currents through the cell membrane can be continued during dilution in order to directly detect the effect of the second ambient medium 5.
  • the ongoing measurement with a rapid exchange of the ambient liquid of the cell 2 is particularly advantageous in the measurement of ligand-activated ion currents.
  • the steps of changing the ambient medium with a running or a repeated measurement of the cell potential according to FIGS. 5 and 6 can be repeated depending on the specific task.
  • the number of repetitions is advantageously virtually unlimited, so that the invention is particularly suitable for the investigation of a large number of test media, in particular in the context of high throughput methods.
  • Advantageously, can be dispensed with the conventional application of test media via hoses, which not only increases the accuracy of the measurement, but also the possible number of applicable test media and thus the measurement throughput can be increased.
  • FIG. 7 shows a further variant of the first embodiment of the liquid holder device 10 according to the invention with the components 11.1, 12.1 and 13 on a patch-clamp pipette 20.
  • the holding element 11.1 is shown in the protruding state as in FIG.
  • the liquid holder device 10 and the patch-clamp pipette 20 are connected to the drive device 50, which for example has a 3-axis Includes manipulator.
  • FIG. 7 shows the swivel joint 13 by way of example at the upper end of the capillary holder 23. Alternatively, the swivel joint 13 can be arranged laterally on the capillary holder 23.
  • the capillary 21 of the patch-clamp pipette 20 is detachably attached to the capillary holder 23.
  • the capillary holder 23 has a suction device 26 for generating a negative pressure, under the action of which the cell 2 can be held on the capillary tip 22.
  • the capillary 21 is inserted as shown in a seal 24, for.
  • the capillary holder 23 with the seal 24 forms a gripper which can be used when pulling the capillary 21 (see FIGS. 11, 12).
  • a conical plug connection can be provided with which the capillary 21 can be exchanged more easily (see FIG. 12).
  • capillaries 21 are used which have a conical shape at their rear end.
  • the holding member is adapted to the liquid for forming the liquid environment at the capillary tip under the action of gravitational force in a vessel, for. B. to provide a cup into which protrudes the capillary in the assembled state of the liquid holder means.
  • the retaining element can not be separated from the capillary tip by the above-described pivoting movement, since otherwise the edge of the vessel would damage the capillary tip. Accordingly, it is provided in the second embodiment of the invention that the holding element is releasably secured to the rod of the liquid holder means.
  • a favorite Buchte variant of the retaining element 11.2 used for the second embodiment of the invention is illustrated in Figures 8A and 8B.
  • the holding element 11.2 according to Figure 8A has the shape of a cup, in the wall of an insertion opening 11.21 is provided.
  • the cup further has a closed bottom 11.23 and a contact groove or a contact gap 11.22, through which an electrode disposed in the insertion opening 11.21 comes into electrical contact with the interior of the cup via the liquid in the cup and the contact channel 11.22.
  • the holding element 11.2 consists z. B. of a plastic that is molded by injection molding. It may be provided, for example, an elastomeric plastic.
  • the rod (not shown in Figure 8A) may be stuck in the insertion opening 11.21 under the action of an elastic tension.
  • a snap-in or latching connection (not shown) may be provided on the insertion opening 11.21.
  • the holding element 11.2 according to FIGS. 8A and 8B has, for example, the following dimensions: outer diameter: 3 mm to 10 mm, cup diameter: 0.3 mm to 3 mm, and axial height 2 mm to 10 mm.
  • Figure 8B illustrates the combination of the retaining element 11.2 with a hollow rod 12.2 (partially shown) which may be advantageously used for liquid transport into or out of the cup.
  • the rod 12.2 has the shape of a tube which is open at its free end on the holding element 11.2 and connected at the opposite end with a liquid reservoir.
  • the rod 12.2 solution can be supplied via the contact gap in the cup or sucked.
  • the provision of the contact gap or the contact channel has the particular advantage that in the tubular design of the rod 12.2 by a pressure change in the rod 12.2, the amount of liquid in the retaining element can be changed.
  • the holding element may be formed as a cup having an open bottom, so that the above-mentioned with reference to Figure IC variant of the first embodiment of the invention results.
  • the holding element 11.2 may have two insertion openings for receiving two bars of the liquid holder device.
  • FIGS. 9 and 10 Various variants of the holding element 11.2 with two insertion openings are illustrated in FIGS. 9 and 10.
  • the rods can be inserted into the insertion openings in a form-fitting or force-fitting manner.
  • the outside diameter of the bars and the inside diameter of the insertion openings are adjusted accordingly.
  • the inner diameter of the insertion openings are shown enlarged for illustration.
  • the liquid holder device 10 shown in FIG. 9A comprises the holding element 11.2 and the rods 12.2, 12.3.
  • the patch-clamp pipette 20 comprises the capillary 21 and the capillary holder 23. Inside the capillary 21 runs the measuring electrode 25, which is connected to a measuring head (not shown in FIG. 9).
  • the rods 12.2, 12.3 of the liquid holder device 10 are fixedly connected (eg, glued or screwed) to the capillary holder 23, so that they are arranged symmetrically on opposite sides of the capillary 21.
  • the rods 12.2, 12.3 extend parallel to the axial extent of the capillary 21.
  • the length of the rods 12.2, 12.3 is selected so that the capillary tip 22 protrudes into the cup of the attached holding element 11.2, without to touch the cup bottom.
  • One of the rods 12.2, 12.3 can be used as an electrode. Accordingly, a contact groove (contact gap) is provided at one of the insertion openings.
  • FIG. 9B shows a modification in which one of the rods (12.3) has a curvature at the end, which serves to fix the retaining element 11.2.
  • the rod 12.3 can be formed of a resilient material and engage in an oblique groove 11.24 on the side of the retaining element 11.2.
  • Figures 9C and 9D show further modifications in which the free ends of the rods 12.2, 12.3 have tips which engage in conically shaped insertion openings of the retaining element 11.2.
  • a lateral screw connection 12.4 of the rod 12.3 with the capillary holder 23 can be provided.
  • the screw 12.4 can be replaced by an annular holder. In this case, there is the advantage that the rods can be easily attached to different types of capillary holders.
  • the preparation of the patch-clamp pipette 20 is carried out.
  • the preparation of the capillary 21 is preferably provided immediately prior to the electrophysiological examination with a pipette pulling device, which will be described below with reference to FIG.
  • the capillary 21 is received by the capillary holder 23.
  • the measuring device 30 is moved with the drive device 50 to a suspension vessel 42. Suspended cells 2 are located in the suspension vessel 42.
  • the patch-clamp pipette 20 is introduced into the cell suspension with the drop-holding device 10.
  • the patch-clamp pipette 20 is aligned so that the capillary 21 extends vertically (perpendicular to the bottom of the suspension vessel 42).
  • the measuring device 30 with the drive device 50 is preferably moved up and down (FIG. 10B).
  • a liquid change takes place in the vicinity of the measured cell 2.
  • This measurement time is determined, for example, as a function of the value of the measured resistance across the cell (so-called "seal resistance") lifted out of the first suspension vessel 42 (FIG. 10C), wherein the holding element 11. stands still and at the capillary tip 22 a liquid environment of the cell 2 is formed.
  • the measuring device 30 is transferred into the further suspension vessel 43 (FIG. 10D), in which a test solution is located. In the test solution, there is a rapid fluid exchange with the liquid in the bowl of the holding element 11.2.
  • the measurement of cell potentials can be continuously continued in order to directly detect the effect of the test solution.
  • the measurement of the cell potential in different test media can be repeated.
  • the amount of liquid in the cup can be changed, which can be shortened by sucking a volume fraction, the dilution time of the solution in the cup.
  • the deposit of cell 2 in a cell culture e.g. be provided at the bottom of a culture vessel.
  • the holding element 11.2 is removed from the capillary 21 (transfer to the release position).
  • the removal of the holding element 11.2 from the rod 12.2 takes place, for example. using a bifurcated tool provided in the culture vessel or in the open air.
  • the measuring device 30 is equipped with a pipette pulling device 70, which is shown schematically in FIG. 11A.
  • a pipette pulling device 70 which is shown schematically in FIG. 11A. Since capillaries for patch-clamp pipettes are very sensitive, easily soiled and therefore hardly transportable, the pipette puller 70 can advantageously be used to fabricate the capillaries 21 of the patch-clamp pipettes 20 at the location of the patch-clamp Measuring device 30 can be used. In particular, an automated operation of the production and recording of the patch-clamp pipette 20 is possible.
  • the pipette pulling device 70 comprises a clamping block 71, a heating wire 72 and a heater 73.
  • a glass capillary with a measuring electrode wire is first inserted into the clamping block 71.
  • the capillary holder 23 is placed on the free end of the glass capillary.
  • the drive device 50, see, for example, FIGS. 5, 6, 10) is preferably used, which thus fulfills a dual function during the production of the capillary 21 and during its transport.
  • the capillary 21 can be withdrawn with the capillary holder 23, so that the desired shape of the capillary tip 22 results. Subsequently, the capillary 21 with the drive device 50 z. B. deposited in a magazine 80, as illustrated schematically in Figure IIB.
  • the magazine 80 includes z. B. a block 81 with holes can be used in the capillaries 21, so that their Kapillarspitzen 22 remain free. The settling takes place with a stripping device or by exerting a pressure pulse in the capillary holder 23.
  • FIG. 12 shows further details of the use of a capillary, in which a conical plug connection 24.1, 24.2 is provided.
  • a capillary 21 is used, at whose ends the plug connections 24.1, 24.2 are fixed by means of screw connections 24.3, 24.4.
  • the screw connections can be replaced by plug connections with at least one sealing ring (see FIG. 13B).
  • the capillary 21 is connected to the plug connections 24.1, 24.2 in the clamping block 71 (see Figure IIA) inserted and pulled out.
  • FIG. 12B shows the finished capillary 21 after removal.
  • FIG. 13 schematically shows a filling device 90 (FIG. 13A), which is provided for filling the capillary 21 of a patch-clamp pipette (FIG. 13B).
  • a working fluid e.g., physiological solution
  • FIG. 13A the filling device 90 illustrated in FIG. 13A is preferably used.
  • Filler 90 includes a fill capillary 91 (e.g., a quartz capillary) mounted in a retainer block 92. Attached to the support block 92 is a connecting tube 93 which provides fluid communication between the filling capillary 91 and a liquid delivery means 94 (e.g.
  • Syringe device forms. Between the liquid reservoir of the liquid conveyor 94 and the holding block 92, there is provided on the outside of the connecting tube 93 a spring means 95 (e.g., a coil spring).
  • the connecting tube 93 is made of a flexible material.
  • the connecting tube 93 and the spring device 95 are elastically deformable.
  • an elastic deformation forces that may arise unintentionally when operating the liquid conveyor 94, recorded, and thus an undesirable power transfer to the capillary of the patch-clamp pipette can be avoided.
  • FIG. 13B shows the combination of the filling device 90 with the capillary 21 of the patch-clamp pipette during the filling process.
  • the capillary 21 is inserted into a conical plug connection 24.5.
  • the plug connection 24.5 (or: receiving device) comprises a conical plug with an axial bore, on the inside of which at least one sealing ring, but preferably two sealing rings 24.6 for holding the capillary 21 are arranged.
  • the axial bore has a funnel-shaped opening 24.7 with a step projection 24.8, which serve the gentle introduction of the filling capillary 91 of the filling device 90 and the formation of a stop for the capillary 21.
  • the wall of the capillary 21 is covered by the projection 24.8, so that the filling capillary 91 is guided by the funnel-shaped opening 24.7 directly into the interior of the capillary 21.
  • connection tube 93 and the spring device 95 avoids excessive stress on the filling capillary 91 and the capillary 21.
  • the introduced filling capillary 91 (as shown in FIG. 13B) to abut the inner wall against the capillary 21 without forming excessive forces.
  • This advantage is achieved because of the defor- mability of the components 93, 95 regardless of the length of the capillary 21, so that the filling device 90 is particularly well suited for the filling of capillaries 21 of different lengths.
  • FIG. 14 shows, by way of example, further details of a capillary holder 23 of a patch-clamp pipette, which can be used to advantage in the implementation of the invention.
  • the capillary holder 23 comprises a capillary electrode 23.1, which is preferably formed by a quartz capillary.
  • the capillary electrode 23.1 is inserted in a carrier 23.2.
  • the carrier 23.2 has at one end a receptacle 23.3 for the capillary (not shown) of the patch-clamp pipette and at the opposite set an electrolyte reservoir 23.5 with an elec- rodenan gleich 23.6 (eg, an Ag-AgCl pellet).
  • the receptacle 23.3 is designed for the direct reception of the capillary 21 or the plug connection 24.1, 24.2, 24.5 (see FIGS. 12, 13) and is preferably equipped with at least one or two sealing rings 23.4.
  • the receptacle 23.3 preferably has a conical form.
  • the capillary electrode 23.1 is preferably a quartz capillary filled with an electrolyte, for example KCl, which opens into the electrolyte reservoir 23.5.
  • an electrolyte for example KCl
  • an Ag-AgCl wire electrode may be provided.
  • FIGS. 11 to 14 may represent independent subject matter, which may be realized with pipettes without the liquid holder device according to the invention described above.

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Abstract

L'invention concerne un dispositif support de liquide (10) conçu pour former un milieu liquide sur une pointe capillaire (22) d'une pipette de patch-clamp (20), ce dispositif comprenant un élément de retenue (11.2) destiné à retenir une goutte de liquide (1), ainsi qu'au moins une tige (12.2, 12.3) servant à positionner l'élément de retenue (11.2) sur la pointe capillaire (22). Selon l'invention, la tige (12.2, 12.3) est conçue de sorte que, lorsque le dispositif support de liquide (10) et la pipette de patch-clamp (20) sont assemblés, l'élément de retenue (11.2) puisse être séparé du capillaire (21). L'invention concerne en outre un appareil de mesure équipé de ce dispositif support de liquide (10), ainsi qu'un procédé d'analyse électrophysiologique d'une cellule biologique au moyen de ce dispositif support de liquide (10).
EP07819499A 2006-11-06 2007-10-31 Dispositifs et procédés pour analyses électrophysiologiques de cellules Withdrawn EP2089508A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102006052231A DE102006052231A1 (de) 2006-11-06 2006-11-06 Vorrichtungen und Verfahren für elektrophysiologische Zelluntersuchungen
PCT/EP2007/009467 WO2008055612A1 (fr) 2006-11-06 2007-10-31 Dispositifs et procédés pour analyses électrophysiologiques de cellules

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EP2089508A1 true EP2089508A1 (fr) 2009-08-19

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EP (1) EP2089508A1 (fr)
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WO (1) WO2008055612A1 (fr)

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US9581562B2 (en) 2011-03-01 2017-02-28 Sophion Bioscience A/S Handheld device for electrophysiological analysis
WO2016054448A1 (fr) 2014-10-02 2016-04-07 Chirp Microsystems Transducteurs à ultrasons micro-usinés piézoélectriques ayant des circuits d'émission et de réception différentiels
CN104774759B (zh) * 2015-04-24 2017-06-13 苏州大学 一种成骨细胞单细胞激励与检测的操作手结构
CN104774758B (zh) * 2015-04-24 2016-11-30 苏州大学 一种成骨细胞单细胞激励与检测的mems系统
CN113406316B (zh) * 2021-06-17 2023-08-08 重庆医科大学附属儿童医院 一种电生理膜片钳灌流装置

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DE19740324C2 (de) * 1997-09-13 2003-05-28 Eppendorf Ag Einrichtung zum Manipulieren von zytotechnischen Instrumenten
US20050241940A1 (en) * 2004-05-03 2005-11-03 Wyeth Fast perfusion system and patch clamp technique utilizing an interface chamber system having high throughput and low volume requirements
SG120998A1 (en) * 2004-09-15 2006-04-26 Offshore Technology Dev Pte Lt Interactive leg guide for offshore self elevating unit
WO2006056920A1 (fr) * 2004-11-26 2006-06-01 Universite De Geneve Systeme de transfert d'echantillons

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