HK1028450B - Positioning and electrophysiological characterization of individual cells and reconstituted membrane systems on microstructured carriers - Google Patents

Description

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This application claims the priority of the Swiss application No 2903/97, filed on 17 December 1997.

Technical field

The present invention relates to a measuring system and a positioning method for cells and vesicles and lipid membranes, which allows studies on membranes, in particular an electrophysiological method for studying channel proteins and receptors coupled to channel proteins or channel proteins via the measured electrical property of the channel proteins. In particular, the measuring method relates to a multiarray patch clamp method, which has the sensitivity and stiffness of the classical patchclamp technique, but also as a result of the inventive method of positioning biological cells or vesicles on microstructured carriers, which allows a more simple production of high-resolution patch signal measurement. The present invention also relates to a positioning method suitable for both electrophysiological and remote sensing.

State of the art

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However, in the context of drug screening, the traditional patch-clamp technique also has major disadvantages: patch-clamp measurements are extremely time-consuming, require specially trained personnel with long experience in this field and are practically not applicable to HTS.

US-A-4 055 799 a method and method for measuring the elastic and dielectric properties of the diaphragm of living cells are known. The apparatus exposed is a container containing two measuring electrodes for voltage differences, two electrodes for the transmission of voltage and current pulses, a separating wall dividing the container into 2 chambers and containing one or more holes, a connector for physiological solution and a connector for the addition of electrolyte solution. For the measurement the cells are partially located in the diaphragm hole so that no planar membranes are formed over the opening. EP-A-094 and WO-A-093 also open through a 50 m × 201 m open tower of a small lens in a carrier, which can be precisely followed by a point of fission or by a position of electric charge (a position defined as the inherent charge of the lens) above an object with no electrical charge.

The present invention was therefore intended to provide a measuring and positioning method which is easy to use and allows for rapid examination, in particular for a (multiarray) patch-clamp method, which has the sensitivity and selectivity of the classical patch-clamp technique but at the same time eliminates its disadvantages due to the inventive method of automatic positioning of biological cells or vesicles with corresponding lipid membranes and the specific surface characteristics of the measuring arrangement.

Description of the invention

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The method of positioning of cells and vesicles or corresponding lipid membranes according to the invention is characterised by the fact that a separating wall of electrically insulating material, hereinafter referred to as a carrier, is placed between two electrodes. The carrier has an aperture and a surface on which the membranes are fixed. The carrier does not have to be made up of a single piece, but may, for example, be made up of a holder on which the material actually relevant for membrane binding and membrane positioning is fixed or may be incorporated into the material, whereby this material has at least one aperture for the binding or membrane positioning of the membranes.It has been shown that cells and vesicles are very well positioned when they are placed as a suspension through an opening of 0,2 to 2 mm in diameter, preferably 0,5 to 1 mm in one electrode or through a tube near the aperture or by means of a pipette. The electrodes above and below the carrier are each given such electrical potentials that the cells show a different electrical potential.The opening of the tube can be of any shape, but is usually elliptical, especially circular, so that it can be arranged, for example, concentrically over the opening.

The fixing of the carrier between the electrodes can be achieved by providing a spacer between the respective electrode and the carrier, which, like the carrier itself, is made of electrically insulating material and has channels located between the aperture and the electrode and in contact with them. Filled with conductive solution, these channels can serve as a reference or sample chamber. It has proven to be advantageous if the sample chamber has a mass of such a size that the reference buffer solution contained in it is fixed by capillary forces.

Since it is useful to contact the membrane with the test solution on both sides, depending on the analysis to be carried out, the addition of a test substance can of course be made from the side which is usually used as a reference side.

The measuring method according to the invention allows, in particular, the measurement of ion channel currents in a reliable and reproducible manner, with a high signal noise distance, based on the precise positioning and subsequent electrically tight binding of vesicles, cells or other biological organs or membranes of the corresponding origin, at microstructured openings (hereinafter referred to as apertures) with a diameter dM < 15 μm, preferably < 10 μm, in particular 0,3 - 7 μm, with special preference for 0,3 - 5 μm and very special preference for 1 - 5 μm. The electrically tight binding of the vesicles or their respective surfaces is achieved by a strong electrical attraction between the surface of the membrane and the membrane.

The method of the present invention has shown that it is advantageous to apply the membrane to a medium as flat as possible. A suitable medium may be composed of various materials; however, the advantage for suitable materials is that they are preferably not only microscopically flat but also relatively flat in molecular terms.

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A particularly suitable medium is a Si/SiO2 or silicon/silicon oxynitride chip, which can be made from commercial Si wavers with an oxide layer of a thickness D usually > 200 nm. Such a medium is easily microstructured. For example, after photolithography, or at apertures of d < 1.5 μm electron beam lithography, the structures can be obtained by anisotropic etching of the silicon in KOH-containing medium as well as reactive ions of the quartz layer. In addition to quartz, layers of glass, solid or gelatinous polymers, etc., also form suitable modified surfaces.

The main features of such structures are the size of the aperture, which should be < 15 μm, usually < 10 μm, especially < 7 μm and preferably < 5 μm, and the size of the window in the surface layer, e.g. quartz, which is preferably < 50 μm, but ideally reduced to the size of the aperture to support a strong focusing of the electric field on the aperture under certain conditions (low buffer conductivity), but above all to reduce mechanical stresses (fracture risk). A strong electric focusing effect corresponding to the strong inhomogeneity of the field (operation with the A to the A vector takes approximately one to one vesper) can be achieved by means of a series of electrical forces (E = A) or a series of electrical forces (E = A) that can be measured in parallel to the force of the electric field.

A planar carrier chip with at least one aperture is placed between two electrodes. Suitable electrodes are, for example, Ag/AgCl, Pt; however, due to their ease of manufacture, Ag/AgCl electrodes are preferred. The electrodes are used not only for voltage clamp but also for positioning vesicles and cells or corresponding membranes.

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The versatility of the measuring device according to the present invention can be further improved by a multiarray design. Microstructuring allows the installation of several apertures in a very small space, which are either coupled to the same electrodes or represent separate measuring compartments, since, for example, Ag/AgCl electrodes are also easily microstructable.

It is also possible to combine the measuring system of the invention with devices for sampling and exchange, sample separation and measurement control, e.g. by connecting the compartments via tubes to a pump system or a device operating by means of hydrostatic pressure differentials or piezo-trophic processes, respectively, the inkjet process or contact transfer process or the electroosmotic process or temperature controlled process or capillary crystallography (CE) or HPLC (High Pressure Liquid Chromatography).

The measurement may be affected by the addition of membrane active substances, e.g. by the addition of pore formers, proteolyposomes and membrane proteins.

The invention is explained in more detail below using the figures.

Brief description of the drawings

Figure 1 is a schematic, non-detailed and non-scale representation of a Si/SiO2 support chip.

Figure 2 is an electron microscopic image of an aperture etched into the SiO2 layer in different views: (A) aperture etched from the SiO2 surface side (B) aperture etched from the Si side (C) overview of the SiO2 side (D) overview anisotropically etched Si side

Figure 3 is a schematic, non-detailed and non-detailed representation of the measuring structure with planar electrodes in the cross section.

Figure 4 is a schematic, non-detailed and non-detailed representation of the measuring structure with point and wire electrodes in the cross section.

Figure 5 shows rhodamine-labeled vesicles (membrane in nature red, light grey in the image) after 24 hours of cleaning, where the number of small vesicles (d < 5 μm) was greatly reduced compared to the uncleaned solution.

Figure 6 shows vesicle-coated surfaces after binding to poly-L-lysine, stripped to very flat forms which do not exhibit carboxyfluorescein fluorescence.

Figure 7 shows a cross-section of fused vesicles, with a calibration bar of 5 μm.

Figure 8 shows a finite element simulation (FEM) of the electric field distribution around a chip with 4 apertures between parallel electrodes. The parameters used are: CPuffer = 10 mM KCl, dApertur = 4 μm and chip gap gap ((4) - electrode ((6.9) = 1 mm. The equipotential lines have a spacing of 4 mV, with the potential difference between the electrodes being 80 mV. The field lines are elliptically distorted (normally circular) in this simulation by the assumption of leakage currents in the periphery of the carrier chip.

Figure 9 shows the time course of vesicle bonding and the formation of a membrane with very high electrical sealing resistance at an aperture of 4 μm (Figure 9A) and 7 μm (Figure 9B), as well as 10 mM KCl, a clamping voltage of -80 mV and a PLL-coated SiO2 surface (PLL = poly-L-lysine bromide).

Figure 10 shows the passage of vesicles through an aperture of 7 μm as modulation in the current-time diagram at a clamping voltage constant VC = -80 mV.

Figure 11 shows in the current-time diagram that the addition of Ca2+ at a final concentration of 4 mM after docking vesicles to the unmodified aperture (7 μm) results in an electrically tight seal between chip surface and vesicle membrane.

Figure 12 shows the time and voltage-dependent switching of alamethicin pores in a membrane produced on the chip (CAlamethicin = 0.1 μg/ml in 85 mM KCl) at negative potentials.

Figure 13 shows changes in membrane resistance of a membrane produced on a Si/SiO2 carrier chip after fusion with vesicles containing nAChR (nicotinic acetylcholine receptor). (A) Random receptor openings in the absence of ligands at 400 mM KCl and positive potentials. (B) No receptor openings are observed (desensitization) 150 seconds after addition of the nAChR agonist carbamycillin (20 μM final concentration).

Ways of carrying out the invention

The method or device (measuring device) of the present invention is particularly suitable for use in drug screening, as a replacement for conventional patch-clamp techniques and as portable biosensors, e.g. for environmental analysis.

The measuring device of the invention has at least two electrodes 6, 9 and separate compartments suitable for the reception of liquid and is characterised by the presence of a carrier 1 between two opposite redox electrodes 6, 9 which enter or touch at least one compartment and are of any shape, containing at least one aperture 3 and separating at least two compartments.

The aperture 3 has a diameter such that, in the presence of a voltage difference across the chip, transmitted by the electrodes 6, 9, an inhomogeneous electric field builds up around the aperture, which becomes considerably larger as the aperture approaches and bends electrophorically near the aperture, cells, cell fragments or biological organelles.which is attractive for biological membranes or has a surface 5 which allows molecular or multi-valent ionic bonding of cells, vesicles, membrane fragments or biological organelles. One such carrier is, for example, a silicon carrier chip with a broken oxide or oxynitride layer. An electrically charged surface 5 may also be produced by modification, in particular by polymerization and/or silanes, e.g. aminosilans, or the carrier may have a coating 2 with an electrically charged surface 5. The carrier 1 may also be partially cleaned and fully hydrophobicised before modifying its surface or before its immediate use in a plastic or oxygen bath.

Due to the specific arrangement, it is not necessary for the measuring arrangement of the invention to have compartments with physical limitations.

In a preferred embodiment of the measuring device, each electrode and at least one aperture 3 in carrier 1 are connected to each other via a channel or chamber 8 in a spacer 7, 10 forming an open or closed compartment.

Also, there may be more than two electrodes 6, 9 and more than one aperture 3 such that at least one electrode, e.g. a reference electrode, is used for measurement over more than one aperture 3, or the measuring device may have a carrier 1 with more than one aperture 3 and twice as many electrodes 6, 9 as apertures 3, such that there is an aperture 3 between each of two electrodes 6, 9.

Furthermore, the compartments may be connected by pipes to a pump system or a device operating on a hydrostatic pressure basis or by a piezo-drop or inkjet process or by a contact transfer process or electroosmotic process or temperature controlled process, so that fluids or samples can be introduced or exchanged into any compartment.

The measuring device of the invention may also be coupled with a sample separation apparatus, in particular capillary electrophoresis (CE) and HPLC, for the analysis of the separated substances, or it may be equipped with means for continuous or periodic testing of the liquid level in the compartments and with means for retesting preset filling parameters.

In another embodiment, the surface 5 of carrier 1 may be structured to give hydrophilic and hydrophobic areas, preferably around the aperture.

Such measuring devices can be used, for example, for the measurements described below:

The following medicinal products are to be used:

The present invention is particularly suitable for the probing of a large number of potential ligands, which can be produced in small quantities by combinatorial chemistry. On the other hand, many receptor proteins, especially ligand-gated and G-protein coupled receptors, are also available in very limited quantities. The method and measuring device of the invention make it possible to work with a very small number of cells, either directly or after previous isolation and reconstitution of the receptor proteins in vesicles or lipid membranes. The uncomplicated arrangement of the sensor elements in arrays allows the simultaneous selection of different sub-substances or receptors.

Replacement of conventional patch-clamp techniques:

As mentioned above, conventional patch-clamp techniques provide the basis for the study of membrane receptor functionality and, in general, of the change in membrane properties in response to signaling and metabolic processes in cells. If isolated cells from a homogeneous cell population are used as the subject of the study, as is often the case, for example, with transformed cells, the method of the present invention can be used as a substitute at least equivalent to the patch-clamp technique.

The following information shall be provided for the purpose of the assessment:

The excellent mechanical stability of the measuring system of the invention and its virtually automatic membrane design allow its use in biosensors. Sensors that are sensitive to very different substrates or metabolites can be built using suitable transformed cells or vesicles of reconstructed receptors or channel-forming proteins. In the case of very good seal formation, as achieved by the device of the invention, the sensitivity is in principle dependent only on the receptor binding constants and can be, for example, in G-protein coupled receptors under a receptor subunit and in ionotropic receptors (e.g. HTN, NACR, GABA, Glucine, Glycine, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR, SCR

The measuring method of the invention is based on the following known measuring principle:

The electrical properties of transmembrane ion channels or ionotropic receptors are generally characterized by so-called voltage-clamp techniques (e.g. classical voltage clamp, patch clamp and oocyte voltage clamp) (see Hamill, Marty et al. 1981 a.a.o.; J.G. Nicholls, A.R. Martin et al. (1992). From neuron to brain: a cellular and molecular approach to the function of the nervous system.

Since the ion flow through ionotropic membrane proteins at 0.1 - 50 pA at VM = -60 mV membrane voltage is generally very small, for an acceptable signal-to-noise ratio the variance of the leakage currents must be about a factor of 5-10 below the signals to be measured.

The problem can be solved in various ways, e.g. by increasing the membrane area to be measured and thus, by summing up, increasing the ion flows. However, in this case, especially in biological systems, specificity is lost.

The problem of sufficient signal noise distance can also be solved by building a very high seal between membrane and electrode. This principle is applied in the present invention. A planar carrier chip with a highly adhesive surface for cells and vesicles is used to do this. This chip separates the two compartments, which are clamped to different potentials during measurement, with a (sub) micrometer-sized opening in the middle. This opening or pore (aperture) is filled with reference buffer solution and is electrically closed during measurement by the strong bonding of cells or vesicles to the surface.

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The critical point in the method of the present invention is the precise positioning of the cells and vesicles above the pore (aperture). This positioning is achieved by the electrode arrangement and field focusing of the invention. The distance between the measuring and reference electrode is usually < 6 mm, but may be greater. The measuring and reference electrodes are each located at a distance of about 0.2 to 3 mm, preferably 0.5-2 mm, in particular 0.5 to 1 mm below and above the carrier chip. A clamping voltage is used to generate an effective electric field for an electrophoretic positioning of the vesicles on the aperture. This voltage is not critical, but is usually in the range of V - 30 mc = 300 mV,The electrophoretic driving force is -60 to -100 mV, and preferably -60 to -80 mV. The resulting electrophoretic driving force causes vesicles and cells to move exactly towards the chip opening following the electric field. Since the field E is highly inhomogeneous and increases considerably in size as the aperture approaches, vesicles are automatically moved towards the aperture. Since the electrophoretic active field strengths are mainly active near the aperture (distance to the aperture < 200 μm), the cells/vesicles must be brought into this area or convectively reached.

It is important for all measurements that the aperture diameter is significantly smaller than the diameter of the vesicles or biological cells (dcell, dVesicle dAperture).

The characteristic electrical properties can be described mathematically as follows:

The thermal noise σ of a circular lipid membrane is proportional to RM-1/2 (B. Sakmann and E. Neher (1983). $\text{σ =}\sqrt{\frac{\text{4}{\text{kTf}}_{\text{c}}}{{\text{R}}_{\text{M}}}}\text{,}$ with RM = Aspect/ (πrM2) it follows: $\text{σ =}{\text{r}}_{\text{M}}\sqrt{\frac{\text{4}{\text{πkTf}}_{\text{c}}}{{\text{R}}_{\text{spec}}}}$

In these formulas σ is the effective flow, r the radius, f the frequency, k the Boltzmann constant, R the resistance and T the temperature.

This results in a consequence for a membrane that can be successfully used for measurement purposes that rM / $\sqrt{{\text{R}}_{\text{spee}}}$ The minimization of this product can be achieved in two ways according to the invention, on the one hand by minimizing the membrane radius rM and on the other hand by additionally sealing the membranes used.

The mechanical stability of the membrane depends on its size. The size of the aperture in the supports determines the diameter of the membrane to be formed. Preferably, the aperture and the window have a comparable diameter. Since the force required to deflect a membrane is proportional to rM-2, structures with dApertur < 5 μm and hence dM < 5 μm result in an extreme increase in membrane stability compared to typical aperture widths of dApertur > 100 μm in conventional BLM systems.

As previously shown, lipid membrane carriers can be made from a variety of materials, but Si/SiO2 and silicon/silicon oxynitride carriers are preferred due to their good and precise machinability.

The Si/SiO2 chips, which are used as the preferred carrier (Fig. 1) for the lipid membranes, can be made from commercial Si wavers 1 with an oxide or oxynitride layer 2 thickness of usually > 200 nm. After photolithography or at apertures 3 of d < 1.5 μm electron beam lithography, the structures are obtained by anisotropic etching of the silicon in KOH-containing medium and reactive ions of the quartz layer.

The main feature of these structures is the size of the aperture, which should be much smaller than the vesicles, cells or organelles used. A reduction in the size of the aperture is advantageous for the formation of a high-quality seal, but on the other hand leads to a reduction in the electrical attraction area around the aperture.

In addition to the machinability, the selection of suitable carrier chip materials must also be made with a view to ensuring that the surface can be modified sufficiently to permit electrostatic or covalent bonding of vesicles or biological cells to it.

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The modification of the carrier surface results in an attraction of vesicles with negative surface charge which is perfectly sufficient for electrically tight junctions between membrane and carrier surface.

Vesicle or cell binding can also be mediated by molecular interactions, e. g. biotin streptavidin or histidine nitrilotriacetic acid (NTA).

Alternatively to the use of polykations described above, the surface can also be modified by other compounds with cationic properties in the desired pH range, such as 4-Aminobutyl dimethyl methoxylan.

Another method for electrically tightly binding vesicles to the SiO2 surface is the addition of Ca2+ ions to the solution, whereby the Ca2+ concentration is increased to > 2 mM after placing a vesicle on the aperture.

A structure with a short distance (D < 5 mm) between the electrodes has been shown to be advantageous for achieving a high field strength for the electrophoretic positioning of vesicles or cells at low voltages (Vc < 200 mV, especially -200 to 200 mV).

The electrodes are preferably placed at such a distance from each other and the compartments and the aperture are filled with buffers or solutions in such a way that field strengths > 100 V/m are obtained in a spherical but arbitrarily shaped area of space entering the compartment fluid around the aperture.

The following describes the specific construction of a measuring system with planar electrodes according to the invention (Fig. 3): an electrode 6, e.g. a silver plate (e.g. purity > 99.98% Ag, but lower purity is also possible) with dimensions of 20 x 20 x 2 mm3, is used as a sensor carrier and at the same time as a reference electrode. On this electrode, by means of a spacer 7, e.g. a 0.5-2 mm thick silicone seal (Sylgard 184, Dow Corning, USA) is placed at the correct distance and parallel to the actual membrane carrier 1. The spacer has a channel of about 1 mm wide and 6 mm long (e.g. a Bench) 8 B, which, for example, is filled with a 90 mm V filter and a reference channel of about 0.8 mm V. For example, the reference channel can be filled with a V filter. For example, after the manufacture of the A/Cl filter, the filter can be placed on a 6 mm V filter and a reference channel of about 8 mm V. For example, the reference channel can be filled with a V filter. For the manufacture of the A/Cl filter, the filter can be placed on a 6 mm V filter and a reference channel of about 1 mm V. For the manufacture of the A/Cl filter, the filter can be filled with a V filter and a reference channel of about 8 mm V. For the manufacture of the A/Cl filter, the filter can be filled with a V filter and a filter with a V filter.

For measurement or membrane production, the measuring solution or vesicle is then fed directly to the aperture 3 or window 4 on the top of the carrier chip 1 or to the top of the measuring electrode 9, e.g. a volume V of about 5-10 μl. To minimise susceptibility to interference, the region around the aperture 3 can be bounded by a silicon ring 10 (Sylgaard) at a distance of, for example, r = 1 mm. This ring 10 together with the meniscus that is exposed between the chip and the upper measuring electrode forms the sample chamber (test compartment). The measuring electrode 9, for example, contains 0.8 μm of highly concentrated chlorinated silver quadratic copper (e.g. Vesicle 4x4 mm), but is positively concentrated in a silicon chip (e.g. 1 mm) at a distance of 11 mm (e.g. 1 mm) from the measuring chip and can be placed in a position parallel to the measuring chip, and the distance between the two is approximately 11 mm.

The system is mechanically very stable despite its openness to liquid storage, by using capillary forces in the filling and storage of the reference and measuring buffer.

Another embodiment uses point or wire electrodes (Fig. 4): the surface modified and, if necessary, fixed on a particularly planar holder, e.g. a glass or Teflon holder, chip 1 is moved between the chlorinated end surfaces 10 of two, if necessary, external (except for the end surfaces) layers with a protective layer 11 above and below chip 1, in particular a Teflon layer, silver wires or silver wire 6, 9 with a diameter of d = 0.1 - 2 mm (protective layer = 2 mm) (distance electrode 6 - electrode 9 - 4 mm). A small electrical tube is placed between the two samples and the chip, or a small hand-held device is placed in the chip, and a small tube or tube is placed in the next tube or tube.

The general measurement system described and the implementation examples described in detail are in principle suitable for integrated systems extended by a sample handling system. These include fluid transport systems which can transport, for example, defined volumes of fluid into and/or out of the fluid compartments of the described structure by means of pumps, hydrostatic pressure differences, electroosmotic, piezoelectric and temperature effects or mechanical displacement. At the same time, a simple parallelisation of the described structure is possible, either on a multi-stage pipe or with several pipes with a single aperture.

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To ensure electrically tight binding of the vesicles to the chip, a net charge of the vesicle surface opposite the carrier surface is required. e.g. the vesicle surface can be negatively charged by palmitoyl phosphatidylglycerol (POPG) to ensure physiological conditions for membrane-embodied proteins as far as possible.

After positioning, cells or vesicles, if they do not burst on their own, can be broken up, e.g. by treatment with hypotonic medium, e.g. pure water.

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According to the invention, a pore-forming agent such as amphotericin B or nystatin can be introduced into the reference compartment after a biological cell or, under special circumstances, vesicles (whose mechanical stability must be sufficiently high) have been bound to the aperture surface, with a significantly higher rate of perforation of the membrane patch across the aperture than with comparable patch-clamp techniques.

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The measurement system and the positioning method according to the present invention have very wide application possibilities. In addition to the possibilities already discussed, they can also be used for the separation or size analysis of vesicles or cells, the positioning of cells for e.g. purely optical examinations or microinjections. The system allows in particular the direct functional analysis of ionotropic membrane proteins, e.g. in ligand binding studies.

The tests may be carried out using cells and vesicles, but also cell fragments, cellular organelles and lipid membranes.

The method allows recording of membrane resistance with good signal-to-noise ratio.

In the method of the invention, the measuring solution or the reference solution or both solutions may be replaced by another solution or a substance to be analysed may be added to the solution on the measuring and/or reference side, e.g. a pore forming agent which may be added to one or both compartments in order to increase the electrical conductivity or permeability of the membrane to certain ions, or proteolyposomes of any size in order to fuse them with the membrane over the aperture 3 and thus make any membrane proteins contained therein accessible for electrical or optical measurements.

The procedure may also be carried out by using apparatus designed to allow access to and performing optical measurements, particularly fluorescence, of the membrane above aperture 3.

Also, several apertures 3 on a support may be used and measurements shall be made sequentially and/or in parallel over at least two apertures 3 and/or in such a way that all or several electrodes on one side of the support 1 have a common electrical potential or are joined together to form one electrode.

The method is now explained in more detail by means of examples which are not to be interpreted in any way as restricting the scope of the invention.

Examples Vesicle formation and size separation

100 μl of azolectin (Fluka) or olecithin (EPC), 50 μl of palmitoyleylphosphatidylglycerol (POPG), 3 μl of dipalmitoylphosphatidylethanolamine-rodamine (DPPE-rodamine) (Molecular Probes, USA) (all 10 mg/ ml in chloroform, Avanti Polar Lipids) and 70 μl of methanol were infused into a film at 400 mmHg in a 10 ml round flask in a rotary evaporator (Rotavapor R-114 book) and then incubated for one hour in a vacuum tube.

The use of purified vesicles to build up electrically tightly bound membranes required the removal of all vesicles and lipid impurities smaller than 10 μm. Inadequate separation resulted in repulsive effects by binding small vesicles near the aperture, which prevented an electrically tight aperture from being formed by large vesicles (> 10 μm). The vesicles were identified for their structural function by a 20 μm lipid membrane analysis at 20 μm. The porosity of the vesicles can be determined by the molecular structure analysis at the temperature of the New York University of Berlin (New York) (Figure 1).

Electrophoresis of the vesicles Option 1: the

Before each measurement, the offset voltage between the electrodes was corrected by applying 5 μl of buffer solution directly to the aperture and then bringing the measuring electrode up to 1 mm nearer the chip surface.

10 μl of a vesicle-containing dispersion was then applied to the top of the measuring electrode, allowing the vesicles to settle through the circular opening in the measuring electrode. Vesicles which had moved through the measuring electrode opening were accelerated directly to the aperture under the influence of the electric field corresponding to the applied electrode voltage VM = -50 to -80 mV. The focusing achieved, measured by the number of vesicle-passages through the aperture on uninfected surfaces, was dependent on the window size (the window is the part of the O2 layer exposed by the A-shaped opening (Fig. 2).

Option two:

Before each measurement, the offset voltage offset between the electrodes was corrected, and after adding 5 μl of buffer solution between the chip and the measuring electrode or reference electrode, the voltage at which the current flow disappears, i.e. I (offset) = 0 pA, was determined.

3 μl of a vesicle-containing dispersion was then added to the measuring compartment near the aperture, whereby, in the case of a plane-parallel electrode arrangement, the vesicles could be sedimented by the circular opening in the measuring electrode.

The electrical positioning described in variants 1 and 2 was superior to the gravitational sedimentation of cells and vesicles also tested in the following points: required number of vesicles or cells, overall membrane formation rate and probability of successful membrane formation or cell attachment.

Vesicle binding and adsorption to modified SiO2 surfaces

The probability of successful and electrically tight positioning was highly dependent on aperture size, SiO2 window size and number, size and size distribution of vesicles. Carrier chips with apertures dApertures < 2 μm and windows < 40 μm, in combination with suspensions of vesicles dViccles > 40 μm, showed a > 90% probability (n > 15 where the number of trials is defined) of electrically tight membrane closures.

The membrane of the vesicles was marked with 0.5% rhodamine (red) and the vesicle lining with carboxyfluorescein (green). The disappearance of all carboxyfluorescein emission (a colour recording shows only the red colour of the rhodamine, which is visible as gray in Fig. 6 as the membrane spots) indicates the release of carboxyfluorescein and thus the bursting of the vesicles and the unilamellarity of these membranes. The very high membrane resistance measured on the carrier chip, which is > 6.4 G (n = 26 RMΩ in 85 mL lipometric KCl, is largely due to the formation of the vesicle, and the polymerization of the vesicle was studied with a JSM 510, a process based on the Fusion and Fusion of polymers.

In an analogue meshesie in symmetrical 10 mM KCl, vesicle binding was measured after appropriate approximation in less than 0.2 sec. with a probability of > 70% (n > 15) and a membrane resistance of RM > 10 GΩ.

Electrical parameters of the lipid membranes

Before each fusion of a vesicle with the modified surface, the resistance of the measuring assembly, determined mainly by the aperture, was determined, which was up to 1 MΩ (usually < 450 kΩ) in 85 mM KCl, depending on the aperture size.

The membranes formed by vesicle fusion or cell bonding over the aperture had a resistance RM > 6.4 GΩ at the same ion concentration, with only a slight change in the capacity of the carrier chips by some pF.

In an analogue experiment in 1 mM KCl, the resistance of the measuring assembly was also determined to be up to 1 MΩ, depending on the aperture size. At the same ion concentration, the resistance of the membranes formed above the aperture was usually RM > 40 GΩ and in 10 mM KCl usually > RM 10 GΩ. The capacity of the carrier chips also changed only slightly in these experiments, from 160 to 280 pF.

The test chemical is used to determine the concentration of the test chemical in the test medium.

In the presence of negatively charged surfaces, such as unmodified SiO2 layers or vesicles fused around the aperture, the passage of vesicles through the aperture was observed by means of resistance changes (Fig. 10).

The passage of very large vesicles with sufficiently fluid membranes, up to 18 sec. long, can be inferred. Especially when using vesicle populations with d > 50 μm (n = 4, where n indicates the number of measurements) and an aperture d = 7 μm, an almost exclusive variation in the passage time was observed at fixed amplitude changes depending on the size of the vesicle.

This method allows the determination of the bulk composition of a solution by analysing the passage time typical of large vesicles (dVesicles >> dAperture) and the change in resistance amplitude typical of small vesicles (dVesicles ~ dAperture).

Observation of alamethycin pores and nicotinic acetylcholine receptors

To verify the biological functioning of the system of the invention, after forming a membrane through the aperture in 85 mM KCl, alamethicin (final buffer concentration 0.1 μg/ml) was given to the measuring chamber (R.B. Gennis, 1989). Biomembranes: molecular structure and function. New York, Berlin, Heidelberg, Springer Verlag). The occurrence of the typical fluctuations of the current (amplitudes and residence times) of alamethicin, corresponding to conductivities of the alamethicin pores of about 600 pS, proves the functional capacity and high sensitivity of the system (Fig. 12).Err1:Expecting ',' delimiter: line 1 column 169 (char 168)In the absence of agonists, typical receptor opening events were observed (Fig. 13A), which largely disappeared within a short time (t < 100 sec) after addition of carbamycollin (20 μM final concentration) (Fig. 13B, desensitisation).

The Binding of Cells

The replacement of the vesicles with biological cells allows them to be positioned and characterised electrically in a similar way to the vesicles used. However, the support of the cell membrane by the cytoskeleton does not automatically cause the cells to burst. This means that after binding the cell to the chip surface, a configuration similar to the Cell Attached technique (CAT, Hamill, Marty et al. a.a.O., 1981) is achieved.

Using CAT, electrical measurements of the entire cell membrane can be performed (e.g. by electrically destroying the membrane patch over the aperture) (Whole Cell Recording).

Furthermore, by cell lysis, single channel events can be recorded in the so-called inside-out configuration, in which the cytosol membrane side is exposed to the test solution.

Claims (30)

1. Measuring arrangement with at least two electrodes (6,9) and separated compartments suitable for the uptake of liquid, characterised in that between two opposite arbitrarily formed redox electrodes (6,9), each one protruding in or contacting at least one compartment, there is an electrically insulating carrier (1) which separates at least 2 compartments from one another and contains at least one aperture (3), wherein the aperture (3) connects the compartments and has such a diameter that, in case of a voltage difference over the carrier mediated by the electrodes (6,9), an inhomogeneous electric field is built up around the aperture, which becomes larger in absolute value when approaching the aperture and, can electrophoretically move near the aperture and towards it vesicles, cells, cell fragments or biological organelles, which makes accurate positioning possible, and wherein an electrically insulating bonding of vesicles, cells, and other biological organelles or organic membranes, respectively, of corresponding origin onto the aperture is made possible.
2. Measuring arrangement according to Claim 1, characterised in that on one side or on both sides of the carrier there are means, which permit an addition of liquid and/or a storage of liquid and/or an exchange of liquid and/or the addition of cells, vesicles, other biological organelles or parts of those between the carrier and electrode.
3. Measuring arrangement according to Claim 1 or 2, characterised in that the compartments, independently of one another have a physical lateral boundary and/or have no physical lateral boundary and the reference volume or the sample, respectively, is fixed in it by capillary forces.
4. Measuring arrangement according to one of the Claims 1 to 3, characterised in that the carrier (1) has an electrically charged surface (5), which is attractive for biological membranes, or has a surface (5) which permits a molecule-specific or multivalent-ion-mediated bonding of cells, vesicles, membrane fragments or biological organelles on it, wherein this bonding is preferably tight enough so that the variance of the occurring leakage currents lies below the signals to be measured of 0.1-50 pA at V_m = -60 mV membrane voltage approximately by the factor of 5-10.
5. Measuring arrangement according to one of the Claims 1 to 4, characterised in that the carrier is a silicon carrier with deposited oxide or oxynitride layer.
6. Measuring arrangement according to one of the Claims 1 to 5, characterised in that the electrically charged surface (5) was produced by modification, in particular by means of polycations and/or silanes, e.g., aminosilanes.
7. Measuring arrangement according to one of the Claims 1 to 6, characterised in that the carrier has a coating (2) with electrically charged surface (5).
8. Measuring arrangement according to one of the Claims 1 to 7, characterised in that before the modification of its surface or before its immediate use, the carrier (1) was cleaned in an oxygen plasma and was made partially or completely made hydrophilic.
9. Measuring arrangement according to one of the Claims 1 to 8, characterised in that one respective electrode and at least one aperture (3) in the carrier (1) are connected to one another through a channel or a chamber (8) in a spacer (7,10) under formation of an open or closed compartment.
10. Measuring arrangement according to one of the Claims 1 to 9, characterised in that the vesicles or cells, respectively, can reach near the aperture by convection or sedimentation.
11. Measuring arrangement according to one of the Claims 1 to 10, characterised in that it has more than two electrodes (6,9) and more than one aperture (3),such that at least one electrode, for example a reference electrode, serves for the measurement over more than one aperture (3)
12. Measuring arrangement according to one of the Claims 1 to 11, characterised in that the carrier (1) has more than one aperture (3) and twice as many electrodes (6,9) as apertures (3), such that there is always one aperture between two respective electrodes.
13. Measuring arrangement according to one of the Claims 1 to 12, characterised in that the compartments are coupled through tubings with a pump system or an equipment that operates on a hydrostatic pressure basis or by means of a piezo drop method or ink jet method, respectively, or by means of a contact transfer method or electro-osmotic method or temperature-controlled method, in such a way that liquids or samples, respectively, can be added to arbitrary compartments or can be exchanged in them.
14. Measuring arrangement according to one of the Claims 1 to 13, characterised in that it is coupled with an apparatus for achieving a sample separation, in particular capillary electrophoresis (CE) and HPLC, and serves for the analysis of the separated substances.
15. Measuring arrangement according to one of the Claims 1 to 14, characterised in that it is provided with means which serve for the continuous or regular checking of the liquid level in the compartments, as well as with means for readjustment of correspondingly preset filling parameters.
16. Measuring arrangement according to one of the Claims 1 to 15, characterised in that the surface (5) of the carrier (1) is structured such that there are hydrophilic and hydrophobic regions, wherein the hydrophilic regions are located preferably around the aperture.
17. Method for the positioning of membranes in the form of cells or vesicles or other biological organelles or of membrane fragments by means of a measuring arrangement according to one of the Claims 1 to 16, characterised in that cells or vesicles or other biological organelles are introduced into the intermediate space between the separating wall or the carrier and electrode, respectively, previously filled with buffer or unfilled and an electrical voltage difference, in particular in the range of -200 mV to +200 mV, is applied between both electrodes, the voltage difference leads to the development of an inhomogeneous electric field and that the electrodes are brought to such a distance from one another and the compartments as well as the aperture are filled with buffers or solutions in such a way that in a spherical but arbitrarily formed space penetrating into the compartment liquid around the aperture, field strengths of > 100 V/m develop, under the influence of which a directed movement of the vesicles or cells towards the aperture takes place.
18. Method for the electrical insulating bonding of vesicles, cells or other biological organelles or membranes or membrane fragments, respectively, of corresponding origin on the aperture, characterised in that the cells or vesicles or other biological organelles are positioned according to Claim 17 and are engaged in an electrically insulating bonding by a strong electrostatic attraction between the carrier surface and the membrane surface.
19. Method for the electrical analysis of natural or artificial lipid membranes, vesicles, cells or biological organelles, characterised in that the membranes are positioned using the method according to Claim 17 or 18, are brought with the surface (5) of the carrier (1) into an electrically insulating bonding above the aperture (3) and permit a recording of the membrane resistance with good signal-to-noise ratio.
20. Method for the electrical measurement of interactions on or in natural or artificial lipid membranes, vesicles, cells, or biological organelles, characterised in that the membranes are produced according to Claim 17 or 18 and that the measuring solution or the reference solution or both solutions are replaced by another solution or that a substance to be analysed is added to the solution on the measuring and/or reference side.
21. Method according to Claim 19 or 20, characterised in that a pore former is added to one or both compartments with the goal to increase the electrical conductivity or permeability, respectively, of the membrane with respect to certain ions.
22. Method according to one of the Claims 19 to 21, characterised in that proteoliposomes of arbitrary size are added to at least one compartment with the purpose of fusing them with the membrane above the aperture (3) and thus to make any arbitrary membrane proteins contained therein accessible to electrical or optical measurements.
23. Method according to one of the Claims 19 to 22, characterised in that after the building up of a membrane above the aperture (3), membrane proteins are incorporated into it.
24. Method according to one of the Claims 19 to 23, characterised in that the membrane located above the aperture (3) is accessible to optical, in particular fluorescence measurements and that these are carried out on it.
25. Method according to one of the Claims 19 to 24, characterised in that a measuring arrangement or a measuring system with several apertures (3) on a carrier is used and that measurements are carried out sequentially and/or parallel over at least two apertures (3).
26. Method according to Claim 25, characterised in that all electrodes on one side of the carrier (1) have a common electrical potential or are combined to one electrode, respectively.
27. Method according to one of the Claims 19 to 26, characterised in that at least one aperture can serve for the measurement of various membrane parameters and that, by means of a pump system, which is connected with tubes to arbitrary compartments or by means of a method based on hydrostatic pressure or by means of a piezo drop method or ink jet method, respectively, or by means of a contact transfer method or electro-osmotic method or temperature-controlled method, liquids or samples are added to or exchanged in arbitrary compartments.
28. Method according to one of the Claims 19 to 27, characterised in that it is performed directly coupled with a sample separation process, in particular capillary electrophoresis (CE) and HPLC, and that it serves for the analysis of the separated substances.
29. Method according to one of the Claims 19 to 28, characterised in that the liquid level in the compartments is checked continuously or regularly and is readjusted corresponding to predetermined filling parameters.
30. Method according to one of the Claims 19 to 29, characterised in that the surface (5) of the carrier (1) is structured in such a way that hydrophilic and hydrophobic regions are obtained wherein the hydrophilic regions are located preferably around the aperture.