EP1349916A2 - Dispositif et procede d'analyse de canaux ioniques dans des membranes - Google Patents

Dispositif et procede d'analyse de canaux ioniques dans des membranes

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Publication number
EP1349916A2
EP1349916A2 EP02718011A EP02718011A EP1349916A2 EP 1349916 A2 EP1349916 A2 EP 1349916A2 EP 02718011 A EP02718011 A EP 02718011A EP 02718011 A EP02718011 A EP 02718011A EP 1349916 A2 EP1349916 A2 EP 1349916A2
Authority
EP
European Patent Office
Prior art keywords
biochip
substrate
opening
cell
channels
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Withdrawn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
EP02718011A
Other languages
German (de)
English (en)
Inventor
Niels Fertig
Jan Behrends
Robert Blick
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.)
Individual
Original Assignee
Individual
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 Individual filed Critical Individual
Priority to EP02718011A priority Critical patent/EP1349916A2/fr
Publication of EP1349916A2 publication Critical patent/EP1349916A2/fr
Withdrawn legal-status Critical Current

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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

Definitions

  • the invention relates to devices and methods for examining ion channels in membranes, in particular devices and methods for carrying out the so-called patch-clamp technique using a biochip, in particular for use in high-throughput methods.
  • Ionic channels are membrane proteins that serve as switchable pores for current flow.
  • ion channels as the smallest excitable biological structures are the fundamental switching elements of the nervous system. Equipping a nerve cell with ion channels of various types therefore essentially determines its role in information processing in the brain. The same also applies to non-neuronal excitable cells, for example those of the heart muscle and its conduction systems. Switching processes in ion channels are examined, among other things, in order to obtain information about possible malfunctions and their remedial action by medication and the like.
  • the patch clamp method is used in the prior art.
  • So-called patch clamp pipettes made of glass are used for this.
  • Such a pipette is shown in FIG. 5.
  • This pipette comprises an opening 59 which has a diameter of approximately 1 ⁇ m.
  • the pipette comprises a pipette shaft 58, in which an electrode 53 is provided.
  • a disadvantage of the known device is that it is not suitable for simultaneously examining a large number of substances or the effect of a substance on a large number of different (for example genetically modified) ion channels.
  • the known device is therefore not suitable for high-throughput tests. As a result, this device can only be used to a very limited extent for substance screening in the pharmaceutical industry.
  • time scale on which the opening and closing mechanisms run in ion channels is only accessible to a very limited extent with this device made of glass pipette, electrode and amplifier.
  • this device made of glass pipette, electrode and amplifier.
  • the bandwidth is limited to below 100 kHz.
  • time scales corresponding to a bandwidth of> 1 MHz would be desirable for examining the opening and closing mechanisms in ion channels.
  • the invention is therefore based on the object of providing a device for examining ion channels in cell membranes which is suitable for high-throughput methods, for example for use in the pharmaceutical industry, and / or which exhibits an improved signal-to-noise ratio and an improved time resolution.
  • a biochip for examining ion channels with a substrate, in which openings in the form of an MxN array are provided for receiving a cell membrane comprising at least one ion channel or for receiving an artificial lipid membrane comprising at least one ion channel, where M> 1 and N> 1.
  • the pipette whose relatively long shaft leads to a high scattering capacity, can be dispensed with. Rather, the critical geometric parameters can be optimized from the outset, as a result of which the signal-to-noise ratio is greatly improved compared to the prior art and the time resolution is accordingly increased.
  • the majority of the openings for receiving membranes that contain ion channels can also be used to parallelize the patch-clamp technique, as a result of which M by N measurements can be carried out simultaneously with a chip to let.
  • this MxN array is particularly advantageous.
  • These cuvette plates can be used in pipetting machines with which substances can be advantageously applied to the biochip described here. It is particularly advantageous that solutions or cells can be removed simultaneously from several cuvettes of the standard cuvette plates and applied to the biochip by pipetting machines or other pipette or cannula arrangements, which are in a rigid arrangement to one another, since the arrangement of the pipettes or cannulas to one another to apply the solutions or cells to the biochip.
  • the biochip according to the invention enables membranes applied to it to be accessible much more easily than the known device. This makes it much easier to observe the membranes, to manipulate them chemically and / or mechanically and / or electrically.
  • the surface in the area of each opening has a device for improving contact with the cell membrane, by means of which improved adhesion of the membrane to the biochip in the area of the aperture (opening) can be ensured. This can also increase the electrical sealing resistance.
  • the device for improving the contact can be designed in the form of a structuring of the surface.
  • the structuring can be provided in the form of one or a plurality of rings which are arranged around each opening, or in the form of one or a plurality of squares or rectangles which are arranged around each opening.
  • the structuring can be provided in the form of a recess concentrically arranged around the opening at a short distance in the surface of the biochip, the diameter of which is a multiple of the diameter of the opening, so that the edge of the opening extends upward from the level of the biochip surrounding it protrudes. In this way, a cell membrane is dented through the edge of the opening, which leads to increased contact between the biochip and the membrane.
  • Each opening can have length and width dimensions that are in a range from 10 ⁇ m to 10 nm. This allows the number of ion channels observed to be set. Furthermore, the membrane area and thus the capacity is reduced by a smaller opening, which leads to a further improved measurement resolution.
  • the biochip according to the invention is also outstandingly suitable for the formation of artificial lipid membranes (artificial lipid double layer) on the opening, in analogy to the known black lipid or lipid bilayer method. This enables the investigation of ion channels by fusion of vesicles containing ion channels with the artificial lipid bilayer.
  • the signal-to-noise ratio can be improved.
  • each opening can be essentially circular. Circular shapes of this type can be easily implemented in the biochip. If a simple implementation is not required, other shapes for the opening cross sections can also be selected.
  • the substrate can have a base section with a first thickness and one or a plurality of have window section or window sections with a second thickness formed in the base section, in which or in each case an opening is provided.
  • the thickness of the base section can be in a range from 1 mm to 100 ⁇ m and the thickness of the window section can be in a range from 1 ⁇ m to 50 ⁇ m.
  • this development can be used to produce apertures with diameters from 10 ⁇ m down to less than 1 ⁇ m with the aid of a dry etching step, laser ablation or etching of a latent ion trace.
  • This further development also enables simplified filling with electrolyte solution and electrical contacting of the aperture.
  • the depression on the underside of the biochip caused by the local thinning enables the easy pipetting in of solutions which penetrate into the aperture by capillary forces and fill it.
  • the substrate can comprise a semiconductor material, such as GaAs, Si, or AlGaAs, or an insulator, such as glass or quartz, or polymers, such as polycarbonate, plexiglass or polydimethylsiloxane (PDMS).
  • a semiconductor material such as GaAs, Si, or AlGaAs
  • an insulator such as glass or quartz
  • polymers such as polycarbonate, plexiglass or polydimethylsiloxane (PDMS).
  • the substrate with the base section and the window sections formed therein consists of one material.
  • the manufacturing process of the biochip can thereby be simplified.
  • a passivating and insulating layer which is applied to the substrate on one or both sides, can be provided.
  • This insulation layer can in particular consist of SiO 2 , Si 3 N, glass or polymers, as well as multilayer systems in which these materials are combined with one another and / or with the above-mentioned semiconductors and / or with metals, and thicknesses of 50 nm to several ⁇ m. With these materials, a sealing resistance of a few G ⁇ , as required for the measurement of currents in the pA range, can be achieved.
  • the insulation layer can also perform the function of an etching stop layer and, in the case of anisotropic etching of the semiconductor, can lead to the formation of a window section in which only the insulation layer is still present.
  • the aperture can then be defined lithographically and introduced into the self-supporting insulation layer by dry etching.
  • polymers such as polydimethylsiloxane (PDMS) can be used as substrate material.
  • PDMS polydimethylsiloxane
  • a 3D negative template (casting mold) which has the inverted structure of the desired biochip is used in the production of the biochip from PDMS described above.
  • the PDMS is initially viscous and, after mixing with hardener, is poured into the mold and cured with or without heating (around 60 to 100 degrees Celsius).
  • the flexible biochip can then be released from the mold, a previous coating of the mold with silanes making it easier to detach.
  • a chemical modification of the surfaces is advantageous for the production of this embodiment.
  • all surfaces of the biochip can have additional insulating and passivating layers made of the materials already mentioned, as well as chemical modifications (silanization, oxidation).
  • electrodes can be provided on one or on both sides of the substrate.
  • electrodes for example made of gold, silver or other suitable metals, can be deposited directly onto the chip. This simplifies the experimental setup, since the electrodes are already firmly integrated on the biochip and therefore there is no need to attach and adjust the electrodes.
  • such an arrangement in particular if the electrodes are brought up to the membrane down to a few ⁇ m, can reduce the parasitic capacitances and resistances even more, which leads to a further improvement in the signal-to-noise ratio.
  • it can be determined whether a biochip with integrated electrodes is used on one or on both sides of the substrate.
  • Ag / AgCl electrodes are particularly suitable as electrodes. These electrodes have the advantage that electrode polarization, which would lead to falsification of the measurement results, is avoided.
  • additional electrodes can be integrated so that high-frequency alternating electromagnetic fields can be applied via the aperture.
  • the dynamics of the ion channels (changes in conformation, ion permeation and binding of ligands) can be influenced or analyzed, in particular, by applying a high-frequency alternating field in the range from MHz to GHz.
  • the use of antenna structures e.g. the bow-tie antenna known from high-frequency technology
  • antenna structures is particularly suitable for creating such high-frequency fields. In this way, an effective coupling of the electromagnetic field to the ion channel can be achieved.
  • An advantageous alternative is the integration of planar waveguides (so-called strip lines) for high-frequency alternating fields.
  • the electrodes can have a width of 40 nm and the electrodes can be brought up to a few nm to the opening in order to optimize the coupling of the power of the alternating fields.
  • Ag / AgCl electrodes in the form of wires or sintered capsules (pellets) can be inserted into this recess be brought in, whereby the aperture is also electrically contacted.
  • interdigital electrodes for generating acoustic surface waves can be provided, with the aid of which cells or liquids can be positioned relative to the aperture of the biochip.
  • surface acoustic waves can keep the cells moving so that they do not adhere to the chip, which would make it impossible to soak them in or otherwise get them into the aperture.
  • electrically and / or optically active and / or passive components can also be integrated on the substrate. This results in a further structural simplification of the test setup.
  • the signal paths can also be kept short, which in turn has a favorable effect on the signal-to-noise ratio.
  • the biochips can have integrated field effect transistor devices for preamplifying measurement signals.
  • the electrodes, the electrically and / or optically active and / or passive components can advantageously be integrated on the substrate, optionally on the etching stop layer or insulation layer.
  • optical near-field devices for observing the ion channel or channels can be provided in all of the biochips described above.
  • the possibility of using near field devices results from the geometry-related easy accessibility of a membrane on the biochip.
  • all scanning probe methods such as atomic force microscopy (AFM), optical scanning near-field microscopy (SNOM) and scanning tunneling microscopy (STM) can be used to observe the membranes without any problems.
  • AFM atomic force microscopy
  • SNOM optical scanning near-field microscopy
  • STM scanning tunneling microscopy
  • Microfluidic channels for on-chip perfusion can advantageously be provided in the biochips described above.
  • a layer of flexible, non-electrically conductive polymer is applied on the receiving side, wherein the layer has at least two openings through which at least the openings are exposed in the substrate.
  • the area of an opening in the polymer layer is thus at least as large as the area of an opening in the substrate.
  • the layer is preferably 10 ⁇ m to 5 mm thick and consists, for example, of PDMS.
  • the openings can be punched, for example.
  • each opening in the polymer layer can expose exactly one aperture and part of the surrounding substrate.
  • several apertures can also be exposed through an opening in the polymer layer; in this case a cuvette spans several apertures.
  • PDMS is particularly suitable as a substrate for these cuvettes, since it has good adhesive properties to glass and quartz as well as to the other substrates mentioned above, from which the biochip can be designed, and is biocompatible.
  • the substrate surface of the biochip can be made hydrophobic by chemical treatment, so that solution drops deposited on the receiving side lie over the apertures with a steep contact angle and remain stably separated from one another. This creates a liquid compartment that is also effective as a cuvette without the aid of another structure.
  • channels in or above the substrate surface parallel to the substrate surface there are channels in or above the substrate surface parallel to the substrate surface.
  • these channels are formed directly as trenches in the surface of the substrate and are then open at the top.
  • the receiving side of the biochip is provided with a layer of PDMS or any other substrate adhering to the biochip which is penetrated by trenches which are open towards the surface of the substrate of the biochip containing the aperture.
  • These trenches can in particular have diameters and depths between 5 and 500 ⁇ m.
  • these trenches are designed such that they run in a cross or star shape towards the apertures and away from there.
  • the dimensions of these channels are dimensioned such that cells move through them either individually (one after the other) or in a different arrangement in a liquid flowing through the channels. Therefore, such channels are suitable for moving cells horizontally to the chip surface from the periphery of the biochip in a targeted manner over the apertures and over them in such a way that the application of negative pressure through an aperture leads directly to the suction of the cell located above them.
  • biochips described above can be produced in a simple manner. Basically, all of the methods have the following steps in common: providing a substrate, forming one or more window sections in the substrate, and forming one opening per window section.
  • the following method is suitable for producing the window section: an insulation layer which is resistant to the wet-chemical etching method (in particular KOH) and which is present on the top and bottom is removed on the underside by a dry etching step in a lithographically defined area, whereby the semiconductor substrate is directly exposed in this area.
  • the following wet chemical etching step (in particular KOH) then causes anisotropic etching to form an etching trench which has the shape of an inverted pyramid.
  • This etching trench can extend to the opposite side if the size of the primary exposed substrate area is sufficient, but in any case remains sealed on one side by the opposite insulation layer, which is resistant to the wet chemical etchant, and thus acts as an etching stop layer.
  • An Si 3 N x layer preferably an Si 3 N layer, an SiO 2 layer or Si 3 N x / SiO 2 multilayer systems have proven to be particularly favorable as the etching stop layer or insulation layer.
  • the opening itself can be formed in the window section by optical lithography and a dry etching step.
  • the opening can be formed, for example, by electron beam lithography and a dry etching step. In a preferred alternative, the opening can be formed by means of a focused ion beam.
  • an isotropic HF etching process can be used to define the window section by locally thinning the glass substrate.
  • the window section can be formed by ablation with a laser of a suitable wavelength or by hot shaping (hot pressing).
  • the actual opening can be made in the window by lithography in combination with a dry etching step.
  • the etching of a latent trace of a single high-energy ion that has passed through the thinned window area can also be used to produce the aperture.
  • the use of an excimer laser with a wavelength in the ultraviolet range is particularly advantageous here.
  • apertures with diameters of less than 10 ⁇ m down to less than 1 ⁇ m can be produced by irradiation with laser light.
  • the substrate surface, the edge of the aperture or the inner wall of the aperture can be treated by local heating, for example with a laser of a suitable wavelength (so-called annealing), to determine the suitability of the substrate surface, the edge of the aperture or to improve its inner wall to form a close contact with a cell membrane, for example to smooth it or to change the chemical structure of the substrate in a suitable manner.
  • local heating for example with a laser of a suitable wavelength (so-called annealing)
  • annealing a suitable wavelength
  • this can also be done by non-local heating of the entire biochip.
  • the temperature reached during local or non-local heating Temperatures can be both below and above the melting point of the respective substrate.
  • the etching step is omitted when the biochip is made from PDMS, since an impression process is used here; i.e. Both the window section and the openings are transferred from a 3D negative template. However, the etching and lithography processes described are used in the production of the negative template.
  • biochips described above can be used in a wide variety of ways, apart from conventional investigations of ion channels in membranes.
  • Portions of the cell membrane of cells can be incorporated into the opening or openings of the biochip.
  • one cell is advantageously first positioned per aperture.
  • individual (not interrelated) cells are applied to the biochip in aqueous suspension, the aperture already being filled with an electrolyte solution.
  • the cells are advantageously applied with the aid of at least one pipette or cannula. This can be done automatically, e.g. by electronically controlled xyz motors. In a preferred development, a separate pipette or cannula is provided for each aperture.
  • these pipettes or cannulas contain integrated electrodes which are suitable for measuring the ion current through ion channels and which are in electrical connection with the cuvette and thus the aperture via the electrolyte solution in the pipette or cannula. This eliminates the need to provide such measuring electrodes on the receiving side on the chip substrate. If, as described above, the biochip is provided with channels running parallel to the substrate surface, one or more individual cells can be flushed into the biochip via these channels and then positioned on an opening in each case.
  • a negative pressure can be applied from the side of the aperture opposite the receiving side, so that the resulting liquid flow moves a cell onto the aperture.
  • a constant electric field can be applied across the aperture, which promotes the formation of a tight connection between the cell and the biochip.
  • direct voltage or alternating voltage fields can be applied via suitable electrodes provided on the biochip, by means of which cells are moved towards the aperture electrophoretically or dielectrophoretically or are held there.
  • surface acoustic waves generated by further electrodes can be used to position liquid drops or cells containing cells on the aperture.
  • cells or other particles or solutions can be added on the receiving side, which due to their specific weight or due to other properties, the cells mechanically and / or by other forces on the receiving side surface of the biochip and / or move the aperture and / or hold it there.
  • All of the described methods for positioning a cell on an aperture are preferably also used to fix the cell on the aperture.
  • the biochips described above advantageously perform an electrophysiological characterization of each cell.
  • contact can also be made via the aperture to the inside of an entire cell.
  • This is advantageously done by abrupt, brief (duration: preferably 10 ms to 10 s, amplitude: preferably -10 to -1000 mmHg) lowering of the pressure in the aperture (suction pulse), by applying an electrical voltage pulse (duration: preferably 0.1 to 1000 ms , Amplitude: preferably 100 mV to 10 V) or by adding a pore former (eg gramicidin or nystatin) to perforate the membrane section located in the aperture.
  • a pore former eg gramicidin or nystatin
  • the presence of a cell above the aperture can be detected by measuring the conductance or the high-frequency impedance or other electrical parameters of the aperture.
  • the suction pulse can then be triggered, for example.
  • active substances are applied or disapplied by flushing or suctioning off solution.
  • the flushing or suction can be done by pipettes or cannulas. If fluid channels are available, they can be used for flushing or suctioning.
  • the application or disapplication of active substances can take place before or during a measurement.
  • all the biochips described can be provided on the underside opposite the receiving side with devices which allow the simple application of a negative or positive pressure relative to the upper side (ie a pressure gradient across the apertures).
  • These can be formed, for example, as liquid-filled hollow chambers located in each opening or window area in a flexible polymer substrate (e.g. PDMS), each of which is connected to the aperture and through it to the top of the biochip, and the volume of which acts from the outside , can be reduced by a mechanical device generated pressure, or increased again by reducing the same.
  • a pressure gradient across the apertures can also be created via microfluidic channels and associated hose systems.
  • one of the biochips described can also be combined with a further, second biochip, which is provided with a device for positioning cells relative to the openings of the first biochip, the respective surfaces on the receiving side being at a fixed or variable distance are opposite.
  • This combination can be done, for example, by a fixed or flexible connection of the two biochips in such a way that their respective surfaces on the receiving side are opposite one another and e.g. are either separated by a gap of 10-1000 ⁇ m in width or lie directly against one another.
  • the device for positioning cells of the second biochip advantageously comprises a device for generating surface waves. If the biochips are mounted directly on one another, the receiving-side surface of the second biochip preferably comprises fluid channels that run parallel to the surface and are open to the surface. In this case, the cells can be flushed in through these fluid channels.
  • the biochips according to the invention can also be used in a measuring probe with a glass tube which is provided on the side of the substrate opposite the side on which the membrane can be applied, the opening of the glass tube facing away from the substrate being designed in such a way that that an electrode can be inserted. In this way, an electrode in ionic solution for examining the ion channel or channels can be brought up to the opening.
  • a holding device made of polycarbonate or another material other than glass, which has a central cavity or a plurality of cavities that communicate with the aperture or the apertures of the biochip and to which the biochip is glued or otherwise is attached and into which an electrode or several electrodes in ionic solution can be inserted.
  • cavities which communicate with the apertures of the biochip, devices can in turn be provided which allow positive or negative pressure to be applied in order to keep cells out of the aperture or to suck them in from a suspension applied on the receiving side.
  • a layer of a flexible polymer substrate for example PDMS
  • this measuring probe can also be integrated without problems in known patch clamp assemblies, in particular in upright and inverted optical microscopes and measuring stations for optical and mechanical scanning probe methods.
  • the opening of the glass tube or the holding device facing away from the substrate can advantageously be designed such that an electrode device can be screwed in.
  • the electrode device can be changed quickly and can also be used again.
  • This arrangement is suitable, inter alia, for a biochip which has integrated electrodes only on the top of the chip.
  • the measuring probe can expediently also be sold together with the screw-in electrode.
  • sealing means for example O-rings, are expediently provided between the opening of the glass tube and the screw-in electrode, so that the electrolyte remains in the glass tube or in the holding device.
  • the glass tube or the holding device is advantageously glued to the substrate or screwable to the substrate with a sealing ring. This ensures a simple and tight connection between the glass tube and the substrate.
  • the screw connection according to the second alternative additionally leads to the simple reusability of the biochip, since it enables the biochip to be cleaned aggressively.
  • the measuring probes described above can advantageously be provided such that they have a device for generating negative pressure in the glass tube or in the holding device.
  • a membrane spot of a cell that is also in solution can be defined with the usual suction technology.
  • everyone can steps required to perform an analysis of ion channels can be performed on a single device. This leads to improved handling of the device.
  • FIG. 1a shows a sectional view of a first embodiment of a biochip according to the present invention
  • FIG. 1b shows a top view of the first embodiment of a biochip according to the present invention
  • 1c shows a plan view of a modification of the first embodiment of a biochip according to the present invention
  • FIG. 2 shows a second embodiment of the biochip according to the present invention
  • FIG. 3 shows a third embodiment of the biochip according to the present invention
  • FIG. 5 shows a pipette for examining ion channels according to the prior art.
  • FIGS. 1a and 1b show a first embodiment 1 of a biochip according to the present invention.
  • This biochip comprises a substrate in which an opening 19 is provided for receiving a cell membrane comprising at least one ion channel.
  • the substrate comprises a base section 10 with a first thickness di and a window section 11 with a second thickness d 2 , in which the opening 19 is provided.
  • the thickness of the base section 10 is in a range from 1 mm to 100 ⁇ m and the thickness of the window section is in a range from 1 ⁇ m to 50 nm.
  • the window section has an area of a few 10 ⁇ m 2 to 0.1 mm 2
  • the opening 19 is essentially circular and has a diameter which is in a range from 10 ⁇ m to 10 nm.
  • the size of the opening depends on how many ion channels in a cell membrane are to be examined.
  • the biochip 1 is formed from a (OOOI) quartz (Z cut) in which the window section 11 is first formed by an anisotropic wet chemical etching step. HF is used as the etchant.
  • this opening is formed by optical lithography and a dry etching step or by electron beam lithography and a dry etching step.
  • the surface of the biochip according to FIG. 1 is provided in the region of the opening with a device for improving the contact between the biochip and the cell membrane.
  • this device is formed by structuring the surface.
  • annular elevations 15, which are arranged around the opening, are provided.
  • FIGS. 1a and 1b The structuring in the biochip according to FIGS. 1a and 1b is only to be understood as an example.
  • other forms of elevations can also be used, for example one or a plurality of squares or rectangles, which are arranged around each opening.
  • Figure 1 c Figure 2 shows a second embodiment of a biochip according to the present invention.
  • This biochip likewise has a substrate 20, 21 in which an opening 29 is provided for receiving a cell membrane comprising at least one ion channel.
  • the geometric shape and the dimensions of the biochip 2 correspond to those of the biochip 1 shown in FIG. 1. To avoid repetition, reference is made in this connection only to the corresponding description of FIG. 1. The reference numerals of corresponding parts differ only in their first digit.
  • the substrate of the biochip 2 comprises a base section 20, which is also formed from quartz, and an etch stop layer, in which the window section 21 is formed.
  • This etch stop layer consists of Si 3 N X ⁇ preferably Si3N 4 .
  • the biochip 2 is distinguished by the fact that it can be produced by a simplified method.
  • etching stop film is thus first applied to the substrate 20.
  • the window section 11 up to the etch stop layer is then formed from the opposite side by an anisotropic wet chemical HF etching step.
  • the opening is formed, preferably by one of the methods described in connection with the first embodiment.
  • FIG. 3 shows a third embodiment of a biochip 3 according to the present invention.
  • the biochip 3 essentially corresponds to the structure of the biochips described in FIGS. 1 and 2, so that here too, reference is made to the description of these chips in order to avoid repetitions.
  • the reference numerals of corresponding parts differ only in their first digit.
  • the base section 30 of the substrate consists of a semiconductor material, for example (100) -Si.
  • An insulating layer is applied to this semiconductor material, in which the window section 31 is formed.
  • the insulating layer 31 also serves as an etching stop layer in the production process.
  • this layer consists of Si 3 N 4 .
  • the insulation and etch stop layer is first applied to the silicon base section 30 using a PECVD method. Then, from the other side, the window portion 31 is formed in the substrate by an anisotropic wet chemical KOH etching step. This is etched through to the etch stop layer. Then, as in the previously described embodiments, depending on the desired size of the opening, this can be formed by optical lithography or electron beam lithography and a dry etching step.
  • electrodes 32 and 33 which in the present case consist of Ag / AgCl, are finally applied to the top and bottom of the substrate.
  • FIG. 3 also shows how a membrane Me with an ion channel I has been introduced into the opening 39.
  • an electrolyte liquid 34 must be provided over the membrane and electrode 32, as well as in the etched trench.
  • FIGS. 1 to 3 merely represent preferred embodiments of the invention and are not to be understood as a limitation thereof.
  • the opening it is not necessary for the opening to be circular. It can have different cross sections depending on the requirements.
  • biochips can also be used to form the biochips.
  • glass can be used instead of quartz and another semiconductor material, for example GaAs, can be used instead of silicon.
  • the surfaces of the substrate can be coated with a passivating layer.
  • Electrodes can also be used, for example those which are suitable for generating an electromagnetic field in the region of the ion channel.
  • electrically and / or optically active and / or passive components can be integrated on the substrate.
  • biochips various methods well known from semiconductor technology can be used to produce the biochips, depending on the materials used in each case.
  • FIG. 4 shows a measuring probe according to an embodiment of the present invention.
  • This measuring probe comprises a substrate with a base section 40 and a window section 41, in which an opening 49 is formed.
  • a first electrode 42 is also arranged on the substrate.
  • a holding device 45 which has a central cavity that communicates with the aperture 49, is attached under the substrate 40, to which an electrode 43 with a holder connects.
  • the measuring probe comprises a device for generating negative pressure in the holding device, which is indicated by reference numeral 46.
  • any of the biochips according to the invention can be used as biochips.
  • the dimensions are determined according to the area of application, in particular the number of channels to be examined.
  • the holding device can be glued to the substrate, for example.
  • the electrode device together with the holder can be designed such that it can be screwed into the holding device from below.
  • a sealing ring can be provided between the opening of the holding device and the screwable electrode.
  • the following describes how ion currents through the ion channel can be measured with the present measuring probe.
  • a cell membrane in electrolyte solution is applied to the substrate.
  • the membrane together with the ion channel is sucked into the opening.
  • electrolyte solution 44 in the measuring probe.
  • the current flowing through the ion channel can be measured via the two electrodes 42 and 43.

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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)
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  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Investigating Or Analyzing Materials By The Use Of Electric Means (AREA)

Abstract

L'invention concerne des dispositifs et un procédé d'analyse de canaux ioniques dans des membranes. L'invention est caractérisée en ce qu'une biopuce dotée d'un substrat comprend des orifices se présentant sous la forme d'une matrice MxN et destinés à loger une membrane cellulaire contenant au moins un canal ionique (I) ou une membrane lipidique artificielle (Me), sachant que M ≥ 1 et ≥ 1.
EP02718011A 2001-01-08 2002-01-07 Dispositif et procede d'analyse de canaux ioniques dans des membranes Withdrawn EP1349916A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP02718011A EP1349916A2 (fr) 2001-01-08 2002-01-07 Dispositif et procede d'analyse de canaux ioniques dans des membranes

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
EP01100458A EP1225216A1 (fr) 2001-01-08 2001-01-08 Appareil pour analyser des canaux ioniques dans un membrane
EP01100458 2001-01-08
PCT/EP2002/000078 WO2002066596A2 (fr) 2001-01-08 2002-01-07 Dispositif et procede d'analyse de canaux ioniques dans des membranes
EP02718011A EP1349916A2 (fr) 2001-01-08 2002-01-07 Dispositif et procede d'analyse de canaux ioniques dans des membranes

Publications (1)

Publication Number Publication Date
EP1349916A2 true EP1349916A2 (fr) 2003-10-08

Family

ID=8176169

Family Applications (2)

Application Number Title Priority Date Filing Date
EP01100458A Withdrawn EP1225216A1 (fr) 2001-01-08 2001-01-08 Appareil pour analyser des canaux ioniques dans un membrane
EP02718011A Withdrawn EP1349916A2 (fr) 2001-01-08 2002-01-07 Dispositif et procede d'analyse de canaux ioniques dans des membranes

Family Applications Before (1)

Application Number Title Priority Date Filing Date
EP01100458A Withdrawn EP1225216A1 (fr) 2001-01-08 2001-01-08 Appareil pour analyser des canaux ioniques dans un membrane

Country Status (4)

Country Link
US (1) US20050009171A1 (fr)
EP (2) EP1225216A1 (fr)
CA (1) CA2434214A1 (fr)
WO (1) WO2002066596A2 (fr)

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DE102017130518A1 (de) 2017-12-19 2019-06-19 ChanPharm GmbH Messgerät, Messverfahren, Hochdurchsatz-Testgerät und Messkit für elektrophysiologische Messungen, insbesondere an Zellaggregaten
WO2021083885A1 (fr) 2019-10-28 2021-05-06 ChanPharm GmbH Appareil de mesure électrophysiologique et procédé de mesure pour acquérir au moins une valeur de mesure électrique au niveau d'un échantillon de cellules biologiques

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DE102017130518A1 (de) 2017-12-19 2019-06-19 ChanPharm GmbH Messgerät, Messverfahren, Hochdurchsatz-Testgerät und Messkit für elektrophysiologische Messungen, insbesondere an Zellaggregaten
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DE102017130518B4 (de) 2017-12-19 2024-04-18 ChanPharm GmbH Messgerät, Messverfahren, Hochdurchsatz-Testgerät und Messkit für elektrophysiologische Messungen, insbesondere an Zellaggregaten
WO2021083885A1 (fr) 2019-10-28 2021-05-06 ChanPharm GmbH Appareil de mesure électrophysiologique et procédé de mesure pour acquérir au moins une valeur de mesure électrique au niveau d'un échantillon de cellules biologiques

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US20050009171A1 (en) 2005-01-13
WO2002066596A2 (fr) 2002-08-29
CA2434214A1 (fr) 2002-08-29
WO2002066596A3 (fr) 2003-03-27
EP1225216A1 (fr) 2002-07-24

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