WO2016026931A1 - Étanchéité fluidique giga-ohmique pour mesures de protéines transmembranaires - Google Patents

Étanchéité fluidique giga-ohmique pour mesures de protéines transmembranaires Download PDF

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Publication number
WO2016026931A1
WO2016026931A1 PCT/EP2015/069145 EP2015069145W WO2016026931A1 WO 2016026931 A1 WO2016026931 A1 WO 2016026931A1 EP 2015069145 W EP2015069145 W EP 2015069145W WO 2016026931 A1 WO2016026931 A1 WO 2016026931A1
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WO
WIPO (PCT)
Prior art keywords
separating element
rings
planar
sub
elements
Prior art date
Application number
PCT/EP2015/069145
Other languages
German (de)
English (en)
Inventor
Eric Nebling
Lars Blohm
Jörg Albers
Original Assignee
Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V.
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 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. filed Critical Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V.
Publication of WO2016026931A1 publication Critical patent/WO2016026931A1/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
    • G01N33/48721—Investigating individual macromolecules, e.g. by translocation through nanopores

Definitions

  • the present invention relates to a sealing system that separates two fluids not only fluidly, but also high impedance from each other.
  • a sealing system is useful for measuring currents in the pico-ampere (pA) region between two liquids, as required, for example, for measuring the functionality of transmembrane proteins that function as ion channels.
  • transmembrane proteins To measure the functionality of transmembrane proteins, they must be incorporated into a thin biological membrane - a double lipid layer.
  • This membrane in turn is usually located in small pores in a carrier substrate, which usually consists of glass or Teflon.
  • a carrier substrate On both sides of this carrier substrate are liquids in reservoirs, which must be separated from each other very high impedance, so as to ensure that the contact between them takes place exclusively over the pores of the carrier substrate or the substances contained therein.
  • the currents to be measured through these pores move in the pA range, so that the electrical insulation of the two liquids from each other, as far as they are not in contact via the carrier substrate, must reach the GigaOhm range.
  • the prior art uses a combination of screw cap, carrier substrate and O-ring or a complete insert.
  • the material suitable for the carrier substrate is not limited to glass or Teflon.
  • silicon chips with nanoporous membranes are also suitable for accommodating the double lipid layer, and thus achieve good results.
  • Such silicon chips are inexpensive to manufacture, so it would be useful to use them as disposable disposable chips in an otherwise reusable device.
  • Such a device would have to be easily disassembled to replace the chip and clean the components.
  • it would have to be designed in such a way that the two liquids in the immediate vicinity of the chip are not only separated from one another fluidly but also electrically, so that the measuring currents can be detected with high accuracy.
  • Such a device could be operated with silicon chips, but possibly also with porous carrier substrates made of other materials.
  • the present invention is directed to the solution of this problem and provides an easily disassembled, fluidly sealing and electrically reliable high-resistance insulating sealing system for a porous, usually planar separator, the sealing system for two separate liquids such a high impedance Resistance that a current flow in the pA range can be reliably determined through the separator.
  • the sealing system according to the invention contains the following components:
  • separating element (5) having pores, which is generally planar at least in its outer edge regions (12),
  • Separating element whose side facing in the direction of the separating element (9, 1 1) is each designed as a planar frame, such that both sub-elements cover the or part of the planar outer edge region of the separating element (5) in all directions,
  • Partial elements is clamped, characterized in that the two O-rings lying in each other, the area in which one of the socket forming the sub-elements (1) covering or part of the planar outer edge region of the separating element, seal, wherein in a relaxed state of O-rings of the inner diameter of the larger O-ring (4) is greater than the outer diameter of the smaller ring (3), while the two O-rings then, when the separating element between the two
  • Part elements is clamped, laterally abut.
  • the inventors have found that the electrical resistance through the remaining air gap between the two O-rings is held securely in the giga-ohm range, even if there are liquids on both sides of the sealing system. This may be due to the fact that the air gap between the rings reliably prevents leakage currents.
  • the use of only one O-ring means that, despite a good fluidic seal, the electrical resistance is not within the desired range because of residual leakage currents.
  • the O-rings are preferably made of a material having a resistance of at least 50 GOhm / mm (gigaohms / mm). Suitable for this example are silicone or Teflon.
  • the wall thickness of the O-rings (viewed in cross-section) is preferably between 0.5 and 2 mm, more preferably about 1 mm.
  • the larger O-ring has an outer diameter of 5 to 20 mm and the smaller O-ring has an outer diameter of 3 to 18 mm.
  • two O-rings with a wall thickness of 1 mm can be used, with the larger of the two O-rings has an outer diameter of 10 mm and the smaller of the two O-rings one
  • the releasable connection means (6) may consist of one, two or more (eg four) clamps or one, two or more than two (eg four) screws which connect the two sub-elements in the region of their frame.
  • the voided separating element (5) which is planar on both sides at least in its outer regions, may be made of any material, e.g. made of glass or Teflon; Preferably, a silicon chip is used. This is thinned in its pore-containing central region in a favorable manner to a thickness of 1 - 5 ⁇ .
  • the sealing system according to the invention is integrally connected to a fluid drum for one of the two fluids.
  • This embodiment is specified in claim 7; it is characterized in that the first sub-element is part of a Fluidgefäßes (1).
  • This fluid barrel has a lateral window opening (8), which is surrounded on its outer side (9) by a planar area.
  • the planar "frame" of the lateral window opening (8) corresponds to the planar frame of the first partial element; the voided separating element (5) covers the window opening and at least part of the planar area in all directions.
  • the second separator is as
  • the invention is illustrated schematically in this embodiment in the accompanying Figure 1, wherein the sealing system in the form shown is in ready state, in which the fastening means, here in the form of two screws 6, the cover 10 against the separator 5 and this pressed against the frame of the window opening 8 of the fluid container 1.
  • the rings 3, 4 are designed and arranged such that there is an air gap 7 between them. This air gap reliably ensures the prevention of any leakage currents. Therefore, when the fluid vessel 1 is filled with a first liquid 1 'and the separator is externally contacted with a second liquid 2, e.g. Membrane channel properties of substances located in the pores of the separating element are detected on the basis of the smallest current flows (ion transport in the pico-ampere range).
  • the rings 3, 4 are in the embodiment shown between the
  • This embodiment can also be designed inversely, namely by placing the separating element on the inside of the window opening and provided from the inside with a cover plate, wherein the releasable connecting means can attack depending on the configuration from the inside or from the outside.
  • the sealing system according to the invention can also be connected to a window of a suitable fluid container. A high-resistance seal for this connection is not required.
  • the sealed fluid container of the present invention may be readily inserted into a second fluid vessel, as needed, into which the second fluid is filled, or may include an integral or fixed second fluid reservoir adjacent the voided separator.
  • both partial elements of the seal are part of a fluid container.
  • This embodiment corresponds to that according to claim 7 with the modification that instead of a cover plate, a second fluid vessel is used which has an identical or similar window with a planar frame as the first fluid vessel.
  • Wall portion such that there can be attached a screw, the e.g. connects a remaining due to the recess web of the wall of this fluid vessel with the container wall of the other fluid vessel.
  • FIG. 1 For this example, an embodiment of the invention as shown in Figure 1 was used.
  • the sealing rings used were two silicone rings each with a wall thickness of 1 mm and outside diameters of 10 and 8 mm. As a separator but no porous
  • Silicon chip used, but a structurally identical chip, in whose membrane there were no pores. This membrane was made of silicon dioxide with a thickness of 4 ⁇ . The chip itself was one-sided (membrane side) completely passivated with silicon dioxide.
  • the fluid container 1 was filled with a phosphate buffered isotonic saline solution (PBS) and placed in a second container which was also filled with this solution. The results of an impedance measurement are shown in FIG. For this purpose, the impedance Z [ohm]
  • the embodiment was repeated with the change that the inner ring was omitted.
  • the result of the impedance measurement is shown in FIG. you detects that the resistance in the low-frequency range was smaller by a factor of 1000 with only 1 megohm.
  • FIG. 1 An embodiment of the invention as shown in Figure 1 was used.
  • the separator used was a silicon chip measuring 10 ⁇ 10 mm (thickness: 0.73 mm).
  • the membrane size was 0.5 x 0.5 mm and in the middle of this membrane were 4 pores (2 x 2), each with 4 ⁇ diameter at a distance of 125 ⁇ to each other.
  • Diph-PC (1, 2-diphytanoyl-3-phosphocholine) in chloroform (10 mg / ml) was carefully applied to the membrane of this silicon chip without directly covering the pores. After drying and rinsing of the chip, it was installed in a device according to FIG.
  • the two chambers of this device were filled with isotonic phosphate buffer (PBS) pH: 7.4, suspended in both chambers freshly chlorinated silver chloride electrodes and the current through the open pores in an electromagnetically shielded measuring chamber in the pA range measured ( Figure 4, 1).
  • the measured values were at 10mV: -0.106 ⁇ ->

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  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Hematology (AREA)
  • Nanotechnology (AREA)
  • Urology & Nephrology (AREA)
  • Food Science & Technology (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Separation Using Semi-Permeable Membranes (AREA)

Abstract

La présente invention concerne un système d'étanchéité qui comprend les composants suivants : un élément de séparation (5) qui est pourvu de pores et qui est plan de toutes parts au moins dans sa zone de bordure extérieure (12), une monture constituée de deux éléments (1, 10) qui est destinée à recevoir l'élément de séparation pourvu de pores et dont le côté (9, 11), orienté en direction de l'élément de séparation, est réalisé sous la forme d'un cadre plan de telle sorte que les deux éléments recouvrent la zone de bordure extérieure plane, ou une partie de celle-ci, de l'élément de séparation (5) sur tous les côtés, deux joints toriques (3, 4) en matériau isolant flexible, et un moyen de liaison amovible (6) destiné à fixer les deux éléments formant la monture l'un à l'autre de telle sorte que l'élément de séparation est serré entre les deux éléments. L'invention est caractérisée en ce que les deux joints toriques, disposés l'un à l'intérieur de l'autre, réalise l'étanchéité de la zone dans laquelle l'un des éléments formant la monture recouvre la zone de bordure extérieure plane, ou une partie de celle-ci, de l'élément de séparation ; lorsque les joints toriques sont détendus, le diamètre intérieur du plus grand joint torique (4) est supérieur au diamètre extérieur du plus petit joint torique (3), tandis que les deux joints toriques ne butent latéralement l'un contre l'autre que lorsque l'élément de séparation est serrée entre les deux éléments. En outre, l'invention concerne un récipient de fluide équipé d'un système d'étanchéité destiné à un élément de séparation qui est pourvu de pores et qui peut être pressé contre une fenêtre latérale du récipient de fluide ; le système d'étanchéité comporte en outre deux joints toriques (3, 4) en matériau isolant flexible qui réalisent, en étant disposés l'un dans l'autre, l'étanchéité de l'élément de réparation poreux plat et soit (a) de la zone plane, ou d'une partie de celle-ci, du récipient de fluide autour de l'ouverture de fenêtre (8) soit (b) de la zone plane, ou d'une partie de celle-ci, de la plaque de recouvrement (10) autour de l'ouverture de cette plaque de recouvrement l'un par rapport à l'autre ; lorsque les joints toriques sont détendus, le diamètre intérieur du plus grand joint torique (4) est supérieur au diamètre extérieur du plus petit joint torique, tandis que les deux joints toriques ne butent latéralement l'un contre l'autre que lorsque l'élément de séparation est serrée entre les deux éléments.
PCT/EP2015/069145 2014-08-21 2015-08-20 Étanchéité fluidique giga-ohmique pour mesures de protéines transmembranaires WO2016026931A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102014111984.3A DE102014111984B3 (de) 2014-08-21 2014-08-21 Fluidische Gigaohm-Dichtung für Transmembranproteinmessungen
DE102014111984.3 2014-08-21

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Publication Number Publication Date
WO2016026931A1 true WO2016026931A1 (fr) 2016-02-25

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110174629A1 (en) * 2008-02-05 2011-07-21 Comm. A L'Energie Atom. et aux Energies Alterna Method for functionalising the wall of a pore
US20130161192A1 (en) * 2011-12-23 2013-06-27 Samsung Electronics Co., Ltd. Apparatus and method for linearly translocating nucleic acid molecule through an aperture
WO2013167952A1 (fr) * 2012-05-07 2013-11-14 The University Of Ottawa Procédé pour l'ajustement de la taille de nanopores à l'état solide
US20140110259A1 (en) * 2011-06-03 2014-04-24 Hitachi High-Technologies Corporation Method and device for optical analysis of biopolymer

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4721157A (en) * 1986-05-12 1988-01-26 Baker Oil Tools, Inc. Fluid sampling apparatus
DE69516459T2 (de) * 1994-07-01 2000-09-21 Bridgestone Corp Vorrichtung zur Schwingungsisolierung

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110174629A1 (en) * 2008-02-05 2011-07-21 Comm. A L'Energie Atom. et aux Energies Alterna Method for functionalising the wall of a pore
US20140110259A1 (en) * 2011-06-03 2014-04-24 Hitachi High-Technologies Corporation Method and device for optical analysis of biopolymer
US20130161192A1 (en) * 2011-12-23 2013-06-27 Samsung Electronics Co., Ltd. Apparatus and method for linearly translocating nucleic acid molecule through an aperture
WO2013167952A1 (fr) * 2012-05-07 2013-11-14 The University Of Ottawa Procédé pour l'ajustement de la taille de nanopores à l'état solide

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
KEYSER U ET AL: "Optical tweezers for force measurements on DNA in nanopores", REVIEW OF SCIENTIFIC INSTRUMENTS, AIP, MELVILLE, NY, US, vol. 77, no. 10, 26 October 2006 (2006-10-26), pages 105105 - 105105, XP012092375, ISSN: 0034-6748, DOI: 10.1063/1.2358705 *

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