WO2007003399A2 - Dispositif d'electrodes, utilisation et procedes de realisation associes - Google Patents

Dispositif d'electrodes, utilisation et procedes de realisation associes Download PDF

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Publication number
WO2007003399A2
WO2007003399A2 PCT/EP2006/006460 EP2006006460W WO2007003399A2 WO 2007003399 A2 WO2007003399 A2 WO 2007003399A2 EP 2006006460 W EP2006006460 W EP 2006006460W WO 2007003399 A2 WO2007003399 A2 WO 2007003399A2
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WO
WIPO (PCT)
Prior art keywords
electrode
tips
base
tip
arrangement according
Prior art date
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PCT/EP2006/006460
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German (de)
English (en)
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WO2007003399A3 (fr
Inventor
Dirk Zimmermann
Ernst Bamberg
Ulrich Zimmermann
Original Assignee
MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V.
Julius-Maximilians-Universität Würzburg
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Application filed by MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V., Julius-Maximilians-Universität Würzburg filed Critical MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V.
Publication of WO2007003399A2 publication Critical patent/WO2007003399A2/fr
Publication of WO2007003399A3 publication Critical patent/WO2007003399A3/fr

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    • 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

  • Electrode assembly its use and method for their preparation
  • the present invention relates to an electrode assembly, its use and - especially galvanic - process for their preparation.
  • the invention has for its object to provide an electrode assembly for electrophysiological examinations of biological species or cells and the like, a process for their preparation and corresponding uses in which access to the interior of a species or cell on especially simple, reliable, gentle and reproducible way to realize.
  • a biological species or cell subsumes a biological cell in the strict sense, a bacterium, a virus, an organelle, a liposome, a vesicle, a micellar structure, their constituents or fragments and their associations or aggregates
  • so-called fusion species or fusion cells should be included. According to the invention, each of these species can be used as a basis for the investigation.
  • an electrode assembly for electrophysiological examination of biological species, particularly biological cells or the like is provided.
  • the electrode arrangement according to the invention is formed with a contact region for contacting the electrode arrangement with at least one biological species, a biological cell or the like. Furthermore, a connection region is formed for external electrical connection of the electrode arrangement.
  • the contact region is formed with an electrode tip or a plurality of electrode tips as electrodes, which extend from the connection region of the electrode arrangement.
  • the electrode tips are each formed with a geometric shape, which in operation an otherwise non-destructive Permeation of the electrode tips into a biological species or cell or the like via the membrane in the interior thereof.
  • the electrode tip or the plurality of electrode tips are suitable for non-destructive penetration of a membrane of the biological species, in particular a biological cell, in order to gain access to the interior of the biological species or cell.
  • the electrode tip or the plurality of electrode tips prefferably be monotonically or strictly monotonically tapering from the connection area. This means, in particular, that the electrode tip tapers uniformly from the proximal to the distal end.
  • the electrode tip or the plurality of electrode tips are each cylindrical or cuboid running from the connection region and at the distal end of the electrode tip or the plurality of electrode tips remote from the connection region with a monotonically or strictly monotonically tapering tip.
  • the electrode tip or a plurality of electrode tips are formed with a cross section which is round, circular, elliptical, rectangular or square.
  • the electrode tip or a plurality of electrode tips are formed with a first and proximal end facing the connection region or forming the connection region.
  • the diameter of the electrode tip or a plurality of electrode tips is formed at the proximal end in the range from about 50 nm to about 5000 nm.
  • the diameter of the electrode tip or a plurality of electrode tips at the proximal end is below approximately 1/10 of the diameter of a species or cell to be contacted.
  • the electrode tip or a plurality of electrode tips are formed with a second and distal end facing away from the connection region.
  • the diameters of the electrode tip or the plurality of electrode tips are formed at the distal end in the range of 1/10 of the diameter of a species or cell to be contacted.
  • the electrode tip or plurality of electrode tips are formed at the distal end with a radius of curvature in the range of about 5 nm to about 50 nm.
  • the radius of curvature of the electrode tip is in particular the radius of that sphere which best approximates the electrode tip at its distal end.
  • the electrode tip or plurality of electrode tips are formed in the region of the distal ends at an acute angle in the range of about 10 ° to about 50 ° as the opening angle of the respective tip.
  • the electrode tip or plurality of electrode tips have a length in the range of about 4/5 of the diameter of a species to be contacted from the connection area.
  • the contact area is formed with a plurality of electrode tips.
  • the electrode tips are formed geometrically equal and / or the same effect.
  • connection area is formed as a materially coherent base with a surface and a bottom.
  • the electrode tip or the plurality of electrode tips are formed extending from the surface of the base.
  • the electrode tip or the plurality of electrode tips extend from the surface of the base vertically or horizontally.
  • Sense vertical extending formed, at least locally.
  • the electrode tips are oriented identically to one another and are designed to be parallel or substantially parallel, at least locally.
  • the electrode tips according to another preferred embodiment of the electrode arrangement according to the invention are alternatively or additionally arranged in the form of a row, matrix or vertical matrix arranged on top of the base.
  • the electrode tips according to a further preferred embodiment of the electrode arrangement according to the invention are formed with the same pairwise distances directly adjacent electrode tips arranged in the main axis directions of their arrangement arrangement.
  • the surface of the base is planar, at least locally.
  • the base and the electrode tip or the plurality of electrode tips are integrally formed with each other as an integral material region.
  • the base and the electrode tips or the plurality of electrode tips are formed integrally connected to one another.
  • the base and the electrode tip or the plurality of electrodes rod tips are formed from the same, in particular electrically conductive material.
  • the electrode tip or the plurality of electrode tips are formed as galvanically grown structures.
  • a carrier having a surface and a bottom side is formed from an electrically insulating material.
  • the proximal ends of the electrode tips and optionally the base are embedded in the carrier and are actually formed below the surface of the carrier and the distal ends of the electrode tips are actually above the surface of the carrier ,
  • the surface of the support is completely or locally conforming and, in particular, running parallel to the surface of the base.
  • the surface of the carrier is completely or locally planar, convex and / or concave.
  • the surface of the carrier is planar or substantially planar and formed with concave depressions in the region of the proximal ends of the electrode tips. It is also possible that the underside of the base is formed on the underside of the carrier at least partially uncovered by the carrier material to allow an external electrical tap.
  • a counter electrode arrangement and / or a reference electrode arrangement are formed electrically insulated from the contact area and the connection area.
  • the counter-electrode arrangement may be formed with one or a plurality of counter-electrodes.
  • the counter electrode arrangement or a part thereof and / or the reference electrode arrangement may be formed on the surface of the carrier.
  • the spatial arrangement and / or the geometry of the counter electrode arrangement are designed to generate a controlled inhomogeneous electric and / or electromagnetic field.
  • the counter electrode arrangement or a part thereof are formed opposite the electrode tip or the plurality of electrode tips.
  • the counterelectrode arrangement or a part thereof can be formed at a distance in the range from approximately 15 ⁇ m to approximately 1 cm from the electrode tip or a plurality of electrode tips.
  • a counter electrode of the counter electrode arrangement is formed with a planar geometry.
  • a counterelectrode of the counterelectrode arrangement has a size and / or area which are large in relation to the size / area of the electrode tips, in particular in a ratio in the range of about 5: 1 or in the range of about 100: 1 or above.
  • the electrode tips and / or the base may e.g. are formed from a material or a combination of materials from the group consisting of silver, gold, platinum, tungsten, alloys, alloys of these metals, platinum-iridium alloys and gold-iridium alloys.
  • a plurality of bases is formed, each with one or a plurality of electrode tips.
  • the bases individually or in groups are electrically isolated from each other and / or spatially separated.
  • a material region is formed with or made of a material or a combination of materials from the group consisting of glasses, glass-like materials, organic polymers and photoresists.
  • the present invention further provides methods for producing the electrode arrangement according to the invention.
  • Process or a combination of processes from the group. which includes galvanic deposition, galvanic growth and galvanic growth.
  • the basic idea is thus the electrical processing and / or manufacturing len 'of the electrode assembly according to the invention.
  • a base electrode e.g. in the form of a base
  • a single electrode tip is formed on the base electrode
  • a base electrode e.g. in the form of a base
  • a plurality of electrode tips is formed on this base electrode.
  • the base electrode or base electrodes with the formed electrode tips may be covered with an electrically insulating carrier region and for the electrode tips to extend with their distal ends above the surface of the electrically insulating carrier.
  • the electrode tips can be acted upon by electrical pulses, in particular in the microsecond range, in such a way that electrical openings are generated in relation to the surroundings.
  • a binding and electrically insulating material is used from the group consisting of covalently binding lipids, covalently binding to metals lipids and insulating plastics.
  • a base electrode is used, in particular in the form of a base, that on the surface of the base electrode or base a structured template is formed such that Recesses in the template Leave areas on the surface of the base electrode or the base at well-defined locations, and then the galvanic process is performed such that exclusively in the region of the recesses of the templates on the base electrode, the electrode tips or preforms thereof are formed.
  • the length of the electrode tips or the preforms thereof is defined by the thickness of the template and the depth of the recesses from the surface to the template to the surface of the underlying electrode or the base. It is also alternatively or additionally conceivable that the thickness of the electrode tips or the preforms thereof is defined by the clear width or the diameter of the recesses in the template.
  • a template with two layers is used, that the layer facing the base electrode or the base has electrically insulating properties and in a well-defibrated manner stable and inert against certain chemical and / or physical effects are formed, and that the second layer or subsequent layers are made removable by relatively mild chemical and / or physical agents, and more particularly by mild chemical reagents.
  • the ratio of the layer thicknesses of the lowest layer of the stencil to the total layer thickness of the subsequent and detachable layers is chosen such that a desired length for the can be reached from the insulating Trager outstanding areas of the electrode tips.
  • the multilayer stencil is first formed on the base electrode or the base, then the galvanic process is carried out, then after termination the galvanic process down to the lowest and layer directly on the base electrode or the base layer all layers of the stencil are removed, and then the lowest and on the base electrode or the base directly resting and remaining layer of the template is used as an insulating support for the electrode assembly.
  • the distal ends of the electrode tips may be post-treated by an electrochemical etching process to sharpen the electrode tip or plurality of electrode tips at their distal ends, particularly in combination with a masking process.
  • the required mask fabrication may e.g. via micropore filters or by treatment of support materials by means of heavy ion bombardment.
  • the invention also provides methods using the electrode arrangement according to the invention and applications of the electrode arrangement according to the invention.
  • the electrode arrangement according to the invention can be used according to the invention for the electrophysiological examination and / or manipulation of a species from the group formed by biological cells, liposomes, vesicles, micellar structures, bacteria, viruses, fusion cells, organelles, genetic, molecular biological and / or biochemical derivatives of which, components of these species and associations of these species.
  • the electrode arrangement according to the invention can also be used according to the invention for the microinjection of a substance into a species from the group formed by biological cells, liposomes, vesicles, micellar structures, bacteria, viruses, fusion cells, organelles, genetic, molecular biological and / or biochemical Derivatives thereof, components of these species and associations of these species.
  • the tip of the electrode tip or the tips of the electrode tips may be charged with the substance to be injected.
  • the application can also be effected by application of electric fields, for example in the case of electrically charged substances, for example DNA.
  • the electrode arrangement is provided embedded in a microstructure.
  • the electrode arrangement is provided in a lap-on-the-chip structure.
  • the electrode arrangement is possible for the electrode arrangement to be provided in or for an assay, in particular for high throughput applications.
  • the species to be examined and / or treated or a plurality of them are directed towards the electrode tip or the plurality of electrode tips.
  • the movement of the species to be examined and / or treated on the electrode tip or plurality of electrode tips is effected by applying force to the corresponding species.
  • the dielectrophoretic force is generated by generating a - in particular high-frequency - inhomogeneous alternating electric field between the electrode tip or plurality of electrode tips and the proposed counter electrode arrangement with the counter electrodes.
  • the electrode tips with an AC voltage in the range of about 10 mV to about 300 V and / or in the frequency range of about 100 Hz or about 60 MHz be applied to generate the dielectrophoretic force.
  • an electrical cell cage is used for the micropositioning of the species during the dielectrophoretic approximation.
  • the cell to be contacted is bulged up by iso-osmolar solutions.
  • the membrane By stiffening reagents, e.g. with EDTA or Pluronium - the membrane can be stiffened and the penetration of the electrode tip facilitated.
  • a counterelectrode 51 of the counterelectrode arrangement 50 has a large and / or flat area that is large in relation to the size / area of the electrode tips 40s, in particular in a ratio in the range of about 5: 1 or Range of about 100: 1 or above, preferably in the range of about 10000: 1.
  • an electrode arrangement in which the electrodes are modified by a chemical reaction such that an electrophysiological examination of biological cells is possible, facilitated or more sensitive, the chemical reaction being mainly an electrochemical oxidation of the aforementioned metals with a halogen, the chemical reaction takes place in particular before or after the contacting of the biological cell, in the latter case
  • an electrode arrangement in which the electrode arrangement is combined with a pressure measuring probe, which is in particular an external pressure measuring probe located outside, or an invasive pressure measuring probe located within a measuring object.
  • electrical isolation of non-cell-contacted free electrodes is performed such that a solution of liposomes of defined size, the minimum diameter being 100 nm and the maximum diameter being 5 ⁇ m, is rinsed across the electrode surface and by applying an alternating current is contacted to the said free electrode tips.
  • a method for electrically contacting a species Z to be examined and / or treated, in particular a biological cell or the like, with an electrode tip 40s of an electrode arrangement 10, in which a patch pipette or patch electrode is used as the electrode tip 40s or the electrode tip 40s and in which the electrode assembly 10 controlled in such a way is applied to an electric field that a dielectrophoretic force on the to be examined and / or treated species Z is exercised in such a way that the species Z to be examined and / or treated approaches the electrode tip 40s and is contacted therewith.
  • the focusing or contacting of the biological cells to be examined electrophysiologically preferably takes place electrically by modulation of the frequencies, the frequencies applied for this being in the range from at least 100 Hz to at most 100 MHz, especially in the range from 100 kHz to 40 MHz.
  • a particular embodiment provides for a combination of the previously described electrode arrangement with a pressure measuring probe, which is an external pressure measuring probe outside or an invasive pressure measuring probe located inside a measuring object.
  • an electrode arrangement in which the electrodes are modified by chemical reaction in such a way that an electrophysiological examination of biological cells is made possible, facilitated or made more sensitive, whereby the aforementioned chemical reaction mainly comprises an electrochemical reaction.
  • cal oxidation of the aforementioned metals with a halogen This chemical reaction can take place before or after the "contacting" of the biological cell, in which case the halogen is obtained from the cytosol of the cell.
  • a possible use is conceivable in which an electrical insulation is not made with cells contacted, free electrodes of the kind that a solution of liposomes of defined size, wherein the minimum diameter 100 nm and the maximum diameter is 5 microns, spooled over the electrode surface and is contacted by applying an alternating current to the said free electrode tips.
  • the dielectrophoretic contacting may also be possible with a construction resembling a normal patch pipette.
  • An electrode - hereafter referred to as A - is surrounded by a micro-glass capillary, which in turn is associated with a physiological
  • the electrode arrangement according to the invention will be synonymously referred to as fakir electrodes.
  • the invention thus also relates in particular to so-called fakir electrodes, their production and their use.
  • electrophysiological techniques the electrical parameters of biological systems can be studied and manipulated. These techniques are applied to cell aggregates, single cells, fragments of cell membranes and liposomes and proteoliposomes, the latter, inter alia, with the help of techniques based on so-called artificial membranes are based). In the following, the spectrum of these biological systems is abbreviated to "cells.” All these electrophysiological techniques have in common that they are also used to investigate the functional properties or for the manipulation of (membrane) proteins and the surrounding membranes.
  • the invention presented here does not have the above-mentioned disadvantages of existing technologies. It is characterized by a high robustness, flexibility in the application and allows both indirect and direct (reversible) electrical discharges to the cells used.
  • the present invention provides, in particular, an electrophysiological measuring arrangement for cells, fusion cells, liposomes, membrane fragments and cell aggregates - in the following simply summarized as cells.
  • the electrical manipulation of the cells is carried out by one or more electrodes that penetrate directly into the cells.
  • the size of the electrodes depends on the cellular system used.
  • the electrode will have a very small diameter for very small cells - diameter in the range of 15 microns, for example in the range of about 900 nm, and only have a small length, for example in the range of about 5 microns. It is also important that the fakir electrodes have a fine tip, for example less than about 500 nm, in order to minimize injury to the cellular system during penetration.
  • FIG. 1 shows a possible electrophysiological arrangement of the fakir technology presented here. Shown is a cell contacted by a fakir electrode with multiple peaks.
  • the carrier material determines the exposed length of the electrode.
  • the fakir electrodes used must have dimensions of the nanometer and micrometer order, both in their geometry and in their length, depending on the cellular system used: the diameter must be between about 50 nm and about 5000 nm, the length between about 500 nm and about 250 microns.
  • the fakir electrodes are made of conductive materials, preferably metals of silver, gold, platinum, tungsten and / or alloys such as Pt-Ir and Au-Ir. production method
  • One possible method of fabricating metal electrodes having the properties described above may be by nanostructuring of electrodes.
  • the surface of a metal electrode can be structured by means of electrodeposition in such a way that it has metal tips with the spatial dimensions described above.
  • different “configurations” are possible, e.g.
  • the latter configuration has the advantage that the individual Fakirspitzen are electrically independent of each other and can be tapped individually.
  • the entire electrode surface (except the fakir tip) must be covered with an electrically insulating layer.
  • an electrically insulating layer For this purpose, two different methods are to be used, a) The first method is based on a coating of the electrode surface with a metal covalently binding lipid. It should be noted that this treatment also stripping the nanostructures on the electrode surface.
  • this treatment also stripping the nanostructures on the electrode surface.
  • electrical ⁇ s pulses that expose the tip, which is done selectively at the points of the nanostructuring and not on the entire planar one (planar in relation to the nanostructures).
  • Electrode surface since the electric fields in the places of greatest curvature are greatest this type can be produced Fakirspitzen with desired geometric and spatial dimensions, b)
  • the second method already intervenes in the manufacturing process during the galvanic deposition for nanostructuring of the surface.
  • This template In order to make a nanostructuring of the surface possible, it must be covered with a stencil.
  • This template has holes. The diameters of these holes determine the diameters of the nanostructures.
  • the layer thickness of the template determines the length of the nanostructures.
  • the stencil used has at least two layers. The lower layer has electrical insulating properties and is stable against chemical / physical agents. The second layer can be dissolved by mild chemical reagents.
  • the layer thickness of the upper layer is chosen so that it corresponds to the desired length of the fakir electrodes.
  • the fakir electrodes presented above should penetrate cells so that they become electrically conductive.
  • Part of the invention presented here is that the electrodes are not brought to the cell as in conventional systems, but the cell to the fakir electrode. This should be achieved by applying a dielectrophoretic force.
  • This force can be generated by the application of high-frequency, highly inhomogeneous alternating fields and, with suitable dielectric properties of the cell - with respect to the dielectric properties of the medium - a migration of the cell in the direction of the Fakirelektrode. It does not end until the fakir electrode is inside the cell. This contacts the cell to the fakir electrode.
  • the described contacting of the cell can also be used for nano- or microinjection of bioactive substances into cellular systems.
  • the fakir electrodes are previously coated or coated with these substances. This can be done, for example, with substances that carry electrical charges (DNA), by applying appropriate electric fields that generate forces on the particles and cause movement to Fakirelektrodenober Structure. If the cell is then contacted with the fakir electrode, then the bioactive substance is in the cell. Advantages of this method are on the one hand the low consumption of bioactive substance, which is used with the "inoculation" of the cell, and the simple selection of the inoculated cells of those, which were not injected, the latter is possible, if one after contacting the cell medium against a cell-free Exchange medium and harvest the daughter cells of the inoculated, contacted cells. " By measuring the electrical parameters In addition, the vitality state of the cells can be determined from the cell contacted, and it is thus possible to optimally control the nutrition of the cells or to stop the harvesting process if the contacted cells lose their vitality.
  • DNA electrical charges
  • Another aspect of the present invention is the use of fakir electrodes for direct or intracellular electrical conduction.
  • the fakir electrode has a very high sealing resistance against the bath solution. This ensures that the resistance (or other electrical parameters of the system) measured by the fakir tip against a reference electrode is determined solely by the conductivity of the "cell" membrane of the cell contacted with the fakir electrode So a very important part of our invention.
  • the fakir electrode (or the fakir board electrode) must be electrically sealed except for the tips of the fakir needles.
  • (2) is described how this is achieved in our invention.
  • Theoretical explanation is based on the fact that under suitably chosen conditions the dielectrophoretic forces only act on objects of specific diameter. With suitable frequencies it is therefore possible selectively to attract smaller sized objects (eg small liposomes of 50 nm to 1 ⁇ m), whereas large objects (eg cells of 20 ⁇ m diameter) do not experience any force.
  • smaller sized objects eg small liposomes of 50 nm to 1 ⁇ m
  • large objects eg cells of 20 ⁇ m diameter
  • Fusion of other cells or liposomes to the system already contacted can also be used to increase the sealing resistance of the fakir electrode.
  • the fusion can be achieved by moderate ⁇ s high-voltage pulses (to merge several laterally or vertically dielectrophoretically arranged cells electrically into a fusion product, so-called electrofusion, thereby simultaneously creating "sensor head" with very large, intact membrane surfaces.
  • the electrical parameters of the cell can be determined by various electrical methods:
  • the fakir electrode in this case should be separated from the cytosol by an intracellular salt bridge.
  • This salt bridge may consist, for example, of hydrogels, such as, for example, alginate, which are doped with Cl-containing salts. Depending on the method, different electrode materials must be used (see (1) and (2)).
  • Fakir electrodes should be tapped together on the one hand and on the other hand individually.
  • the use of multiple fakir electrodes has the advantage that the failure of one or more electrodes is reduced by e.g. a possible deposition of cytoplasmic lipid and protein components or of membrane components during penetration due to the redundant system can be compensated.
  • the advantage of several independent faded Fakirelektroden has the additional advantage that in parallel several different cells can be tapped simultaneously and thus when using extremely small volumes of solution many independent results can be determined.
  • a further measure for increasing the mechanical stability of the complex of Fakir electrode and contacted cell is the embedding of this complex (the hybrid sensor head) in a cross-linked hydrogel matrix (for example, of a Ba 2+ ions is crosslinked Alginatmatrize). This immobilization of the complex simultaneously ensures long-term vitality of the complex and also facilitates the (cryo) preservation of the hybrid sensor heads.
  • the transmembrane resistance an important electrical parameter of the cell
  • the transmembrane resistance depends on the ion channels in the membrane of a system whose electrical conductivity is specifically influenced by a broad spectrum of analytes (ligands, inhibitors, etc.). can be.
  • the fakir technology can be used in screening tools (eg high throughput drug target method).
  • targets eg membrane proteins such as ion channels, see above
  • Such hybrid sensor heads formed from fakir electrodes and contacted cells allow the screening of a wide range of drugs in analytical laboratories ("high throughput screening", “lab on the chip") as well as under in situ conditions (as " In addition to native cells, animal and plant sensor cells should be used which can be tailored by specific heterologous overexpression of transporters or cell-cell or cell-membrane fusion.
  • Specifically designed sensor heads can be made available as disposables for universal electronic peripherals
  • the sensor units can be manufactured either individually or in the form of micromodules, comparable to microtiter plates
  • the latter configuration guarantees the possibility of redundant measurements with comparable sensor heads under identical measuring conditions a se hr high reliability of the analysis.
  • complex detection of multiple components in small sample volumes eg for the purpose of drug screening, can be carried out with high accuracy.
  • the sensor head must be integrated into a probe - lab-in-the-probe - which provides direct, minimally invasive access to liquid-filled compartments of plant or animal / human systems.
  • the new sensor head technology is to be connected to a miniaturized tube / pressure sensor / catheter system.
  • the integration of the sensor head / catheter assembly into a measuring machine based on the principle of a belt hole punch is also planned.
  • An important requirement for being able to contact cells successfully is the shape of the tip of the electrode tip 40s and in particular its radius or radius of curvature Ks at the distal end 40d of the electrode tip 40s, which should not exceed 1/10 of the diameter Dz of the cell Z to be penetrated.
  • the cell membrane M is stretched, that is, the cell Z is filled. This can be achieved by using non-isoosmolar solutions in which the cells Z are incubated or used as measuring media 30.
  • the correct dielectrophoretic force is generated, matching the diameter Dz of the cell Z and the distance of the cell Z from the fakir electrode 40s.
  • the parameters for this process are chosen for each cell type depending on the above conditions. They are in the specified ranges. Not necessary, but favorable is the application of a modulated alternating field, ie an electric field, which changes in a pre-programmed manner during the attraction experiment.
  • the time range for generating the attractive force is about 10 ⁇ s to about 30 s.
  • the modulation of the alternating field can be effected via the amplitude-lowering of the amplitude, for example as a ramp protocol, in particular linear or exponential-or via the frequency.
  • the dielectrophoretic force is inversely proportional to the fifth power of the distance between the cell Z and the fakir electrode 40s.
  • the attraction process is such that initially, by choosing appropriate frequencies and high amplitudes, a relatively small force is generated on cell Z.
  • the force rapidly increases and the cell Z can be drastically accelerated if the original field parameters are maintained. This can lead to too fast movement of the cell and destruction of the cell Z, e.g. by bursting, lead in the contact.
  • too low attraction forces cause the cell Z not to be penetrated by the fakir electrode 40s, because then the mechanical resistance of the membrane M of the cell can not be overcome.
  • the automated use of the fakir electrode in mechanical systems is to be achieved in that the chip carrying the fakir electrodes can be inserted into a microfluidic chamber.
  • this chamber is to ensure that cells can be positioned automatically and with regard to the individual fakir tips exactly opposite the fakir electrodes. This is to ensure that the system can be contacted with automatically applied dielectrophoresis protocols - as already described.
  • the microfluidic system should also allow the possibility of change of solution.
  • FIG. 1 is a schematic and sectional side view of a first embodiment of the electrode assembly according to the invention with an electrode tip.
  • Fig. 2 is a schematic and sectional side view of another embodiment of the electrode assembly according to the invention having a plurality of electrode tips.
  • 3A, 3B are schematic and sectional side views of another embodiment of the electrode assembly according to the invention, once with and without a contacted biological cell.
  • FIGS. 4A-4D are schematic and sectional side views of various further embodiments of the electrode arrangement according to the invention.
  • FIGS. 5A, 5B illustrate certain details of the invention in the form of a schematic and sectional side view and a schematic plan view, respectively, of an embodiment of the electrode arrangement according to the invention.
  • FIG. 6 is a schematic plan view of another embodiment of the electrode arrangement according to the invention.
  • FIG. 7A is a schematic and sectioned side view of another embodiment of the electrode assembly according to the invention.
  • FIG. 7B is an electron micrograph of an embodiment of the electrode arrangement according to the invention, which has been galvanically produced according to the invention.
  • FIGS. 8-10 show, in the form of microscopic photographs, certain applications which are suitable for the electrode arrangement according to the invention.
  • FIGS. IIA-12B schematically illustrate further applications of the present invention.
  • Fig. 1 is a schematic and sectional side view describing a first embodiment of the electrode assembly 10 according to the invention and its application in the investigation of a cell Z.
  • the embodiment of the electrode arrangement 10 according to the invention shown here is based on a carrier 20 or carrier substrate 20 with a surface 20a and a bottom 20b.
  • a contact region 4OK in part and the connection region 4OA are formed integrally in the support 20 in such a way that the electrode tip 40s forming the contact region 4OK of the electrode assembly 10 with its terminal region 40AA or proximal end 40p completely below the surface 20a of the support 20 and with its distal end 4Od, which faces away from the connection area 4OA, is formed strictly above the surface 40a of the carrier 40.
  • connection region 40A is formed by a base 40b which has a one-piece material region - here in the form of a planar plate - realized whose upper side 40ba is contacted with the proximal end 40p of the electrode tip 40s and the bottom 40bb flush with the bottom 20b of the carrier 20 and thus allows an external contact.
  • an electrical tap into the interior I of a contacted cell Z takes place in that the distal end 40d of the electrode tip 40s penetrates through the cell membrane M into the interior I of the cell Z and so on the conductivity of the electrode tip 40s realized a corresponding electrical tap.
  • a current measurement or voltage measurement can take place via the outer measuring circuit 60 and the connection lines 61 and 62, so that charge carriers which have been displaced by the transmembrane protein P can be measured as corresponding displacement currents I (t) as a function of time, the electrode tip 40s being referred to as
  • the first electrode of the electrode assembly 10 and provided in the surface 20 a reference electrode R are formed as a corresponding second measuring electrode, wherein the circuit is closed by the correspondingly to be envisaged aqueous measuring medium 30.
  • the reference electrode R can serve as a measuring electrode. It is also conceivable, however, for this reference electrode R to be used for the approximation and contacting of the cell Z with the contact region 40K by forming a counter electrode 51 of a counterelectrode arrangement 50.
  • the counterelectrode arrangement 50 can also have a counterelectrode 51, which supports the electrode tip. ze 40s of the contact area facing, as indicated by a dashed line.
  • FIG. 1 The embodiment of FIG. 1 is defined with only a single electrode tip 40s in the contact region 4OK.
  • the contact region 40K of the electrode arrangement 10 is defined by a plurality of, in particular identical or identically acting, electrode tips 40s.
  • FIG. 2 shows such an embodiment with a plurality of similar electrode tips 40s in the contact region 4OK.
  • the embodiment of the electrode arrangement 10 according to the invention shown here is based on a carrier 20 or carrier substrate 20 with a surface 20a and a bottom 20b.
  • a contact region 4OK in part and a connection region 4OA are again formed integrally in the carrier 20 in such a way that the electrode tip 40s forming the contact region 4OK of the electrode assembly 10s is completely below the surface 20a of the terminal 40A facing or proximal end 40p Carrier 20 and with its distal end 40d, which is oriented away from the terminal portion 4OA, strictly above the surface 40a of the carrier 40 is located.
  • connection region 4OA is likewise formed by a so-called base 40b, which realizes an integral material region whose upper side 40ba is in contact with the proximal end 40p of the electrode tip 40s and whose underside 40bb terminates flush with the underside 20b of the carrier 20 and thus again an external contact allowed.
  • Electrode tips 40 s penetrate through the cell membrane M into the interior I of the cell Z and so realize a corresponding electrical tap on the conductivity of the electrode tips 40s as an electrode.
  • a current measurement or voltage measurement can take place via the outer measuring circuit 60 and the connection lines 61 and 62, so that charge carriers displaced by the transmembrane protein P can be measured as corresponding displacement currents I (t) as a function of time, the electrode tip 40s being the first electrode the electrode arrangement 10 and a reference electrode R provided in the surface 20 a is designed as a corresponding second measuring electrode, the circuit being closed by the aqueous measuring medium 30 to be correspondingly provided.
  • the reference electrode R can again serve as a measuring electrode. However, it is also conceivable that this reference electrode R is used for the reflector-approximation and contacting of the cell Z with the contact region 4OK by forming a counter-electrode 51 of a counter-electrode arrangement 50. Alternatively or additionally, the counter-electrode arrangement 50 can also have a counter electrode 51, which faces the electrode tips 40s of the contact region, as indicated by a dashed representation.
  • FIGS. 3A and 3B only differs from the embodiment shown in FIG. 2 in that the surface 20a of the carrier 20 does not run strictly planar, but in the region of the electrode tips 40s form a concave depression 22, in particular in the form of a depression, so that, as becomes clear in the transition from the state of FIG. 3A to the state of FIG. 3B, an approaching cell Z better abuts against the surface 20a in the region of the recess 22 can cling to the surface, so that better Abdichtwiderstande at the points X over the proposed measuring medium 30 to avoid short circuits are possible.
  • FIGS. 4A to 4D show a schematic and sectional side view of various embodiments of the electrode arrangement 10 according to the invention.
  • a single electrode tip 40s is provided which defines the contact region 40K of the electrode assembly 10 and which is attached and contacted at its proximal end 40p to the top surface 40ba.
  • the electrode tip 40s and the base 40b as a connection portion 40A are formed einstuckig.
  • a single and separate electrode tip 40s which is to form the contact region 40K of the electrode assembly 10 can also be attached to the upper side 40ba of the base 40b in a downstream process, so that gives a one-piece structure, as shown in Fig. 4B.
  • FIG. 4C likewise shows a one-piece embodiment of the electrode arrangement 10 according to the invention, but this time with a plurality of electrode tips 40s, which are each formed with their proximal ends on the upper side 40ba of the carrier 40b.
  • Fig. 4D again a Ausbowungs form of the inventive electrode assembly 10 is shown in which there is no Einstuckmaschine between the electrode tips 40 and the base 40 b. Rather, the electrode tips 40s, which are the contact region 4OK of the electrode assembly 10 of FIG. 4D form on the top 40ba applied in a downstream process, electrically and mechanically contacted.
  • the embodiment of the electrode arrangement 10 according to the invention which is illustrated in the form of a schematic and sectional side view or in the form of a schematic plan view in FIGS. 5A and 5B, shows a plurality of electrode tips 40s in a row on the base 40b in the form of a planar Plate are arranged, in a non-integral manner.
  • the distal ends 40d and the proximal ends 40p of the electrode tips 40s are shown, which face away from the upper side 40ba of the base 40b and are in contact therewith.
  • the electrode tips 40s shown in Figs. 5A and 5B have a length Ls and are equidistant from each other in pairs with equal distances dd, ds. Also their geometric design is the same. This means that they have the same square cross-section with an edge length Dp and a corresponding diameter Dp in the region of the distal end 40p.
  • the electrode tips 40s are of equal length and extend strictly monotonically tapering to their tip.
  • FIG. 6 shows an embodiment of the electrode arrangement according to the invention, in which a plurality of electrode tips 40s, which form the contact region 40K of the electrode arrangement 10 according to the invention, are arranged in the form of a square matrix with an equal spacing dd, ds from each other and with an identical diameter Dp, which here describes the diameter of the circular cross-section proximal end 40p of the respective electrode tips 40s.
  • FIG. 7A shows an embodiment of the electrode arrangement according to the invention, in which a type of lawn of a plurality of electrode tips 40s is provided on the base 40b of the electrode arrangement 10.
  • FIG. 7B is an electron micrograph of an embodiment of the electrode arrangement 10 according to the invention, which has been galvanically produced according to the invention.
  • the individual electrode tips 40s do not have the same length, and have different orientations and directions.
  • FIGS. 8 to 10 show microscopic photographs of corresponding applications of an electrode arrangement 10 according to the invention with a single electrode tip 40s, which is in contact with a test cell Z.
  • carrier substrate 20a surface, surface area, top

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Abstract

L'invention concerne un dispositif d'électrodes (10) pour effectuer des examens électrophysiologiques de cellules biologiques (Z) et autres. Ce dispositif d'électrodes (10) comprend une zone de contact (40k) pour mettre en contact ledit dispositif d'électrodes (10) avec une cellule biologique (Z) ou autre, ainsi qu'une zone de raccordement (40A) pour le contact électrique externe du dispositif d'électrodes (10). La zone de contact (40K) est constituée par une pointe ou par une pluralité de pointes d'électrodes (40s) qui s'étendent à partir de la zone de raccordement (40a) et ont une forme géométrique permettant, en fonctionnement, une pénétration sans endommagement dans une cellule biologique (Z) ou autre, jusqu'à l'intérieur de la cellule (I) en passant par sa membrane (M).
PCT/EP2006/006460 2005-07-01 2006-07-03 Dispositif d'electrodes, utilisation et procedes de realisation associes WO2007003399A2 (fr)

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DE200510030858 DE102005030858A1 (de) 2005-07-01 2005-07-01 Elektrodenanordnung, deren Verwendung sowie Verfahren zu deren Herstellung

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DE102008009826A1 (de) 2008-02-19 2009-08-20 Max-Planck-Gesellschaft Vorrichtung zur Bestimmung eines elektrophysiologischen Parameters von biologischem Zellmaterial, Verfahren zur Herstellung einer Messelektrode für eine derartige Vorrichtung sowie Verfahren zur Vermessung einer Zelle mithilfe einer derartigen Vorrichtung
DE102012002459A1 (de) * 2012-02-08 2013-08-08 Universität Rostock Elektrophysiologische Messanordnung und elektrophysiologisches Messverfahren
EP2631393A1 (fr) 2009-05-19 2013-08-28 Pacadar S.A. Tour pour éolienne

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DE102007019842A1 (de) * 2007-04-25 2008-10-30 Forschungsinstitut Für Die Biologie Landwirtschaftlicher Nutztiere Verfahren und Anordnung zum elektrischen Kontaktieren eines membranumhüllten Objekts mit einer Elektrode
CN110669664B (zh) * 2019-10-22 2024-06-21 南方电网科学研究院有限责任公司 一种硅橡胶表面藻类的电场处理试验方法

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DE102008009826A1 (de) 2008-02-19 2009-08-20 Max-Planck-Gesellschaft Vorrichtung zur Bestimmung eines elektrophysiologischen Parameters von biologischem Zellmaterial, Verfahren zur Herstellung einer Messelektrode für eine derartige Vorrichtung sowie Verfahren zur Vermessung einer Zelle mithilfe einer derartigen Vorrichtung
EP2631393A1 (fr) 2009-05-19 2013-08-28 Pacadar S.A. Tour pour éolienne
DE102012002459A1 (de) * 2012-02-08 2013-08-08 Universität Rostock Elektrophysiologische Messanordnung und elektrophysiologisches Messverfahren
DE102012002459B4 (de) * 2012-02-08 2015-06-25 Universität Rostock Elektrophysiologische Messanordnung und elektrophysiologisches Messverfahren

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