WO2007107375A2 - Pipette en verre ou capillaire en verre pour expériences de patch-clamp - Google Patents

Pipette en verre ou capillaire en verre pour expériences de patch-clamp Download PDF

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Publication number
WO2007107375A2
WO2007107375A2 PCT/EP2007/002587 EP2007002587W WO2007107375A2 WO 2007107375 A2 WO2007107375 A2 WO 2007107375A2 EP 2007002587 W EP2007002587 W EP 2007002587W WO 2007107375 A2 WO2007107375 A2 WO 2007107375A2
Authority
WO
WIPO (PCT)
Prior art keywords
glass
pipette
diameter
tip
capillary
Prior art date
Application number
PCT/EP2007/002587
Other languages
German (de)
English (en)
Other versions
WO2007107375A3 (fr
Inventor
Albrecht Lepple-Wienhues
Original Assignee
Flyion Gmbh
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 Flyion Gmbh filed Critical Flyion Gmbh
Publication of WO2007107375A2 publication Critical patent/WO2007107375A2/fr
Publication of WO2007107375A3 publication Critical patent/WO2007107375A3/fr

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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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/02—Burettes; Pipettes
    • B01L3/021—Pipettes, i.e. with only one conduit for withdrawing and redistributing liquids

Definitions

  • the invention relates to novel glass pipettes or glass capillaries for patch clamp experiments and to the use of glass pipettes or capillaries for patch clamp measurements. Finally, the invention also relates to patch clamp measurements with glass pipettes or capillaries themselves.
  • lipid membrane As is known, the interior of living cells is surrounded by a lipid membrane, which seals off the processes inside the cell from the external environment. These lipid membranes are largely impermeable to charged particles. For this special transport proteins, which are embedded in the membranes, take over the transport of the charged particles (ions) through the membrane. These transport proteins thus form the basis for a large number of physiological functions.
  • the patch-clamp technique allows the direct measurement of currents generated by the ion transporters in the membrane with high temporal resolution.
  • the resulting benefits are known. For example, it is possible to determine directly the effect of signal molecules or pharmacologically active substances on a target protein.
  • the patch-clamp technique allows to precisely control the electrical and chemical environment of a membrane and also allows the application of signal molecules, pharmacologically active substances, etc. on both sides of a membrane.
  • the patch-clamp technique is known to the expert and is widely used, it should be dispensed with further basic explanations here.
  • the usual procedure in the patch-clamp technique is to mechanically approximate a glass capillary or glass (micro-) pipette of a cell and thus of its cell membrane and to fix it there by suction. This leads to an electrically dense connection between the cell membrane and the tip of the pipette.
  • This electrically sealed connection is a prerequisite for a high-resolution low-noise measurement of small and very small currents.
  • the electrically sealed connection is often referred to as "seal.”
  • a so-called “gigaseal” is required, ie, an electrically sealed connection in which the electrical resistance reaches the gigaohm range.
  • resistors of 10 G ohms and above are needed to assure that even small ions such as protons do not pass uncontrollably between cell membrane and glass surface.
  • the glass pipettes or glass capillaries used for prior art patch clamp experiments typically have a cone-shaped tip and a portion adjacent to this tip that is substantially tube-shaped.
  • Such pipettes or capillaries are usually made by conventional glass blowing technique, namely by pulling (pulling) glass tubes after or at the melting of the area where the conical constriction and tip is to be generated.
  • Such pipettes or capillaries with cone-shaped tips are then used both for those techniques. applies, in which the glass capillary is introduced from the outside to the cell membrane, as well as in those techniques in which the cell is fixed inside the capillary to form a gigaseal.
  • the invention has the object of further improving patch-clamp techniques in which a cell or a corresponding biological structure is fixed inside a pipette or capillary to form a gigaseal.
  • patch-clamp techniques can be used even better for investigations in which rapid mass transport or mass transfer inside the pipette or capillary at the investigated membrane is required.
  • the glass pipette or glass capillary according to the invention has a cone-shaped tip to which a substantially tube-shaped
  • the pipette / capillary is designed so that its diameter is along a length L which is less than 150 ⁇ m. extends, widened from a diameter Di of less than 50 microns at the base surface of the tip to a diameter D 2 of at least 100 microns in the tubular portion.
  • a cone is known to be a structure that has the shape of a cone or truncated cone. Accordingly, in the invention, the pipette / capillary has a tip with the shape of such a cone. Due to its manufacturing process (usually pulling / pulling from a capillary), this tip is not geometrically exactly conical in shape, but can essentially be described by such a cone shape. Since the tip is necessarily open, d. H. is not closed, it will be in the case of the invention is usually a cone in the form of a truncated cone.
  • a truncated cone is a body of revolution that results from cutting a smaller cone from a straight circular cone parallel to the base surface.
  • two parallel circular surfaces are created, of which the larger is referred to as the base surface and the smaller one as the cover surface. If this is again transferred to the case of the present invention, the cone of the tip with its (larger) base surface adjoins the tubular portion at the pipette / capillary.
  • the decisive feature of the pipette / capillary according to the invention is that its diameter over a short distance (length L of less than 150 microns) of a diameter D 1 at the base surface (less than 50 microns) to a diameter D 2 in the tube-shaped Extended section (at least 100 ⁇ m).
  • L length of less than 150 microns
  • D 1 at the base surface less than 50 microns
  • D 2 in the tube-shaped Extended section at least 100 ⁇ m
  • cannulas and capillaries which generally have a diameter of at least 100 .mu.m, can be inserted as far as possible into the pipette / capillary according to the invention.
  • the substances mentioned can be brought very close to the cell membrane of the cell fixed to the base surface.
  • it can be prevented in this way that solution residues remain in the region of this cell membrane when the solution changes.
  • the length L is less than 50 ⁇ m, preferably less than 30 ⁇ m. Accordingly, in these cases, the diameter of the pipette / capillary extends along this length from a diameter Di to a diameter D 2 .
  • the length L is less than 10 ⁇ m, preferably approximately only 5 ⁇ m.
  • the cell or corresponding biological structure in which the cell or corresponding biological structure is fixed inside a pipette or capillary, as small as possible biological structures should be fixed to form a gigaseal. Accordingly, it is preferred according to the invention if the diameter Di at the base surface of the tip is less than 20 ⁇ m, in particular less than 10 ⁇ m. With such small diameters then also the mentioned small biological structures can be fixed. For this purpose, reference may also be made to the disclosure of WO 02/10747 A2.
  • the diameter D 2 in the tubular section is at least 150 ⁇ m, preferred at least 200 microns, is.
  • cannulas with corresponding diameters can be introduced into the interior of the pipette or capillary according to the invention for bringing substances to the cell membrane. This facilitates handling and thus the corresponding patch clamp measurements.
  • tubular portion of the pipette or capillary according to the invention over its length usually will not have a constant diameter D 2 .
  • D 2 the diameter in the tubular section generally has at least the stated value at all points.
  • the cone of the tip extends from its top surface (opening of the tip) over as short a distance as possible to its base surface.
  • This can also be described in such a way that in the region of the tip of the pipette / capillary a possible blunt cone is aimed at.
  • this can be represented by the so-called cone angle. This is the angle that remains when you cut a sector out of a circular piece of paper so that the paper can be turned into a cone. The larger the remaining cone angle, the more "blunt" the cone will be.
  • the cone of the tip should have a large cone angle, preferably a cone angle of at least 240 °.
  • the pipette or capillary according to the invention can be made of all glass materials which are used in the performance of patch clamp experiments.
  • the glass is an aluminosilicate glass or, more preferably, a borosilicate glass.
  • the wall thickness of the pipette / capillary according to the invention is basically not critical, as long as patch-clamp experiments can still be carried out in which the cell is fixed inside the capillary to form a gigaseal.
  • the total length of the pipette / capillary according to the invention is likewise not critical. It is preferably between 10 mm and 50 mm, in particular about 25 mm.
  • the pipettes / capillaries according to the invention are produced, in particular, by extracting (stuffing) cone-shaped tips from tubular capillaries by melting.
  • a gas pressure is applied to the interior of the capillary after the area of the tip has been softened by melting.
  • the invention further includes the use of a glass pipette or glass capillary having a cone-shaped tip and a substantially tubular portion adjacent to the tip for performing patch-clamp experiments involving a cell or similar structure into the inner lumen of the glass pipette is introduced and positioned there in the region of the tip under formation of a gigas seal between the cell membrane and the inner surface of the capillary.
  • the diameter of the pipette of a diameter of less than 50 microns at the base surface of the tip expands suddenly, ie over a small length to a larger diameter, in particular to a diameter of at least 100 microns in the tubular portion.
  • a glass pipette or glass capillary according to the invention described above is preferably used.
  • the invention comprises a method for measurements on cells or similar structures according to the patch-clamp technique, in which at least one cell is introduced into the inner lumen of a glass pipette or glass capillary and in the region of the tip of the glass pipette to form a gigaseal between cell membrane and Inner surface of the glass pipette is positioned.
  • the glass pipette / glass capillary has a cone-shaped tip and an adjoining the tip substantially tubular portion.
  • the pipette / capillary is designed such that the diameter of the pipette of a diameter of less than 50 microns at the base surface of the tip jump-shaped, d. H. extended over a small length, in diameter, in particular to a diameter of at least 100 microns in the tubular portion.
  • the described method is preferably designed such that the glass pipette / glass capillary is an inventive pipette / capillary described above.
  • Fig. 1 shows the form of a pipette or capillary according to the invention in a schematic representation.
  • a glass capillary with a cone-shaped tip is produced from a thin glass capillary made of borosilicate glass by melting and drawing.
  • This glass capillary (glass pipette) is transferred into a device, with the aid of which this capillary can be melted in the region of the cone and pressurized inside with a gas pressure. In this way, the glass capillary shown schematically in FIG. 1 is provided.
  • the glass capillary 1 has a substantially cone-shaped tip 2 and a substantially tubular portion 3 adjoining this tip 2.
  • the cone of the tip has a diameter Di, which in the present case is about 40 ⁇ m is.
  • the diameter of the capillary extends to a diameter D 2 of about 140 microns.
  • the diameter of the capillary in the tubular portion increases with increasing distance from the base surface of the cone.
  • Decisive for the present invention is the sudden expansion of the diameter of the capillary from the diameter Di to the diameter D 2 along a length L of less than 150 microns, in the case illustrated along a length L of about 12 microns.
  • a patch-clamp experiment can be carried out in an advantageous manner, as it is explained in WO 02/10747 A2.
  • the cell to be examined is in the area of the base surface of the tip (diameter Di) under training fixed on a gigaseal.
  • cannulas or capillaries having a diameter of, for example, 100 ⁇ m can be introduced into the region of the fixed cell in a simple manner. Accordingly, a rapid change of solution and a rapid introduction of active substances or potential active substances to the cell membrane is possible.

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  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • Biochemistry (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Devices For Use In Laboratory Experiments (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

L'invention concerne en premier lieu une pipette en verre ou un capillaire en verre (1) pour réaliser des expériences de patch-clamp, cette pipette ou ce capillaire présentant une pointe (2) conique et une section (3) sensiblement tubulaire adjacente à cette pointe (2). Ladite pipette ou ledit capillaire se caractérise en ce que son diamètre passe d'un diamètre D1 inférieur à 50 µm au niveau de la base de la pointe à un diamètre D2 supérieur ou égal à 100 µm dans la section tubulaire sur une longueur L inférieure à 150 µm. L'invention concerne en outre l'utilisation de cette pipette ou de ce capillaire pour réaliser des expériences de patch-clamp, ainsi que des mesures de patch-clamp au moyen de pipettes ou de capillaires de ce type.
PCT/EP2007/002587 2006-03-23 2007-03-23 Pipette en verre ou capillaire en verre pour expériences de patch-clamp WO2007107375A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE200610014513 DE102006014513A1 (de) 2006-03-23 2006-03-23 Glaspipette oder Glaskapillare für Patch-Clamp-Experimente
DE102006014513.5 2006-03-23

Publications (2)

Publication Number Publication Date
WO2007107375A2 true WO2007107375A2 (fr) 2007-09-27
WO2007107375A3 WO2007107375A3 (fr) 2007-11-22

Family

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2007/002587 WO2007107375A2 (fr) 2006-03-23 2007-03-23 Pipette en verre ou capillaire en verre pour expériences de patch-clamp

Country Status (2)

Country Link
DE (1) DE102006014513A1 (fr)
WO (1) WO2007107375A2 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012155973A1 (fr) 2011-05-19 2012-11-22 NMI Naturwissenschaftliches und Medizinisches Institut an der Universität Tübingen Procédé et dispositif pour la détermination automatique de la position d'un microsystème pour la manipulation d'un micro-objet sphérique

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001048475A1 (fr) * 1999-12-24 2001-07-05 Astrazeneca Ab Dispositif et procede permettant de faire des mesures electriques sur un objet
WO2002010747A2 (fr) * 2000-07-31 2002-02-07 Flyion Gmbh Technique et appareil patch-clamp permettant d'effectuer des mesures sur des cellules

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001048475A1 (fr) * 1999-12-24 2001-07-05 Astrazeneca Ab Dispositif et procede permettant de faire des mesures electriques sur un objet
WO2002010747A2 (fr) * 2000-07-31 2002-02-07 Flyion Gmbh Technique et appareil patch-clamp permettant d'effectuer des mesures sur des cellules

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HAMILL O P ET AL: "IMPROVED PATCH-CLAMP TECHNIQUES FOR HIGH-RESOLUTION CURRENT RECORDING FROM CELLS AND CELL-FREE MEMBRANE PATCHES" PFLUEGERS ARCHIV: EUROPEAN JOURNAL OF PHYSIOLOGY, SPRINGER VERLAG, BERLIN, DE, Bd. 391, 1981, Seiten 85-100, XP000196663 ISSN: 0031-6768 *
NEHER E ET AL: "THE EXTRACELLULAR PATCH CLAMP: A METHOD FOR RESOLVING CURRENT THROUGH INDIVIDUAL OPEN CHANNELS IN BIOLOGICAL MEMBRANES" PFLUEGERS ARCHIV: EUROPEAN JOURNAL OF PHYSIOLOGY, SPRINGER VERLAG, BERLIN, DE, Bd. 375, Nr. 2, 1978, Seiten 219-228, XP009075888 ISSN: 0031-6768 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012155973A1 (fr) 2011-05-19 2012-11-22 NMI Naturwissenschaftliches und Medizinisches Institut an der Universität Tübingen Procédé et dispositif pour la détermination automatique de la position d'un microsystème pour la manipulation d'un micro-objet sphérique
US11054409B2 (en) 2011-05-19 2021-07-06 Multichannel Systems Mcs Gmbh Method and device for automatically determining the position of a microsystem for manipulating a spherical microobject

Also Published As

Publication number Publication date
DE102006014513A1 (de) 2007-09-27
WO2007107375A3 (fr) 2007-11-22

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