EP2577301A2

EP2577301A2 - Method for producing a bilipid layer, and microstructure and measuring assembly

Info

Publication number: EP2577301A2
Application number: EP11727916.6A
Authority: EP
Inventors: Jan Behrends; Gerhard Baaken
Original assignee: Albert Ludwigs Universitaet Freiburg
Current assignee: Albert Ludwigs Universitaet Freiburg
Priority date: 2010-06-07
Filing date: 2011-06-01
Publication date: 2013-04-10
Also published as: DE102010022929B4; JP2013534619A; WO2011154114A3; US20130140192A1; WO2011154114A2; DE102010022929A1

Abstract

The invention relates to a method for producing a bilipid layer above a microcavity (104), comprising the following steps: filling the microcavity (104) with an electrolyte solution (110); moving a fluid (112) containing dissolved lipids in a first direction (114) toward the microcavity (104); moving the fluid in a second direction (118) away from the microcavity (104); monitoring the formation of the bilipid layer above the microcavity by detecting an impedance between a counter-electrode (122) connected to the fluid and a measuring electrode (120) which is disposed in the interior of the microcavity (104).

Description

METHOD FOR PRODUCING A BILIPIDATE LAYER AND MICROSTRUCTURE AND MEASURING ARRANGEMENT

The present invention relates to a method for facilitating the production of a bilipid layer over a microcavity that is open on one side, as well as to a microstructure and a measuring arrangement that enable this type of simplified and facilitated production of lipid bilayers.

Synthetic bilipid layers are interesting for research and industry for many reasons. They are models of cell membranes in which the biological functions of reconstituted membrane proteins can be investigated with particular precision. Since, with the help of this model system, currents through individual ion channels in membranes were measured shortly before the development of the so-called patch-clamp technique, it is experiencing an increasing renaissance in recent years. One reason for this is the successful miniaturization of systems for the production of and measurement on such bilipid layers [see, for example, US Pat. B .: Wonderlin, W.F .; Finkel, A .; French, R.J., Biophys J, 1990 58, (2), 289-297; Akeson, M .; Branton, D .; Kasianowicz, J.J .; Brandin, E .; Deamer, D.W., Biophys J, 1999 77, (6), 3227-3233; Pantoja, R .; Sigg, D .; Blunck, R .; Bezanilla, F .; Heath, J.R., Biophys J, 2001 81, (4), 2389-239; Pantoja, R .; Nagarah, J. M .; Starace, D.M .; Melosh, N. A .; Blunck, R .; Bezanilla, F .; Heath, J.R., Biosens Bioelectron, 2004 20, (3), 509-517; Ready, N .; Meyer, C .; Blick, R. H .; Trautmann, C; Behrends, J.C. Phys. Rev E, 2001 6404, (4); Ready, N .; Klau, M .; George, M .; Blick, R. H .; Behrends, J.C., Appl Phys Lett, 2002 81, (25), 4865-4867; Ready, N .; Blick, R. H .; Behrends, J.C., Biophys J, 2002 82, (6), 3056-3062; Mayer, M .; Kriebel, J.K .; Tosteson, M.T .; Whitesides, G.M., Biophys J, 2003 85, (4), 2684-2695; Malmstadt, N .; Nash, M.A .; Purneil, R. F .; Schmidt, J.J., Nano Lett, 2006 6, (9), 1961 -1965; Sondermann, M .; George, M .; Ready, N .; Behrends, J.C., Bba-Biomembranes, 2006 1758, (4), 545-551; Baaken, G .; Sondermann, M .; Schlemmer, C; Rest, J .; Behrends, J.C. Lab Chip, 2008 8, (6), 938-44]. In addition to a significantly increased measurement resolution in terms of time and amplitude, on the other hand, this miniaturization opened up the perspective of a greatly increased throughput of measurements per time due to parallelization.

From the demand side, the renaissance is due to at least two factors, namely the need for channel and channel pharmacological drug screening To investigate transporter proteins that are not or hardly accessible for the patch-clamp technique (in particular proteins in membranes of intracellular organelles and the increasing use of bacterial pores as molecular coulter counter and nanoreactors for single molecule analysis (eg mass spectroscopy of polymers or DNA sequencing by Oxford Nanopore Technologies).

However, the potential of the miniaturized bilipid layers for high-throughput investigations has not yet been sufficiently exploited technically. Such as B. in the dissertation Baaken, Gerhard: "Development of a Microsystems Platform for Parallel Studies of Ion Channels in Artificial Cell Membranes", Albert-Ludwigs- University Freiburg, 2008, have been carried out in recent years with the development of chip-based planar patch Clamp technologies set new standards in terms of miniaturization and integration density for highly parallel measurements on cell membranes, but above all a simple and reproducible and easily automatable solution for the production of double lipid layers was lacking for high-throughput investigations on synthetic membranes The newly developed method for the automated production of bilipid layers on microstructured cavities makes it possible in a very simple way to adapt existing devices for high-throughput electrophysiology to a complete system in a short time for fully automated, parallel measurements on bilipid layers. In summary, the method to be presented here overcomes fundamental practical difficulties and disadvantages with regard to the generation of free-standing lipid bilayers, which are of interest for answering fundamental questions in electrophysiology and for pharmacological drug screening.

Previous solutions have above all the disadvantages explained below. An essential step towards the automation of electrophysiological studies on cell-physiological model membranes in general, and further on membrane proteins, is to develop a method by which the production of free-standing lipid bilayers over (small) apertures can be reproducibly uncomplicated without intervention by the experimenter can be realized. For the application of lipid bilayers to a surface or via an aperture, two basic methods have been established in the last thirty years: The Painting technique, see, for example, Müller et al., Z Kreislaufforschung, 1963, 52 (7) 534 ff.), And the Langmuir-Blodgett technique.

The coating technique is extremely simple to apply, but its success depends almost entirely on the experimenter's manual dexterity, and therefore automation of this method seems very difficult.

Application by the Langmuir-Blodgett technique and methods developed or related to it is widely used in the transfer of defined mono- or multimolecular layers to a substrate surface. The authors of the article Takagi, M.A., K .; Kishimoto, U., Annu. Report Biol. Works Fac. of sei. Osaka Univ, 1965 13, 107-1 10, and later the authors of the article Montal, M .; Mueller, P., Proceedings of the National Academy of Sciences, 1972 69, (12), 3561-3566, simplified this technique so that it is used today in many experiments for the preparation of bilipid membranes, as described, for. In the article Daneion, C; Lindemann, M .; Borin, C; Fournier, D .; Winterhalter, M., IEEE Transactions Nanobioscience, 2004 3, (1), 46-48. Basically, in this method, first an aperture in a thin hydrophobic substrate film, which separates two compartments, wetted from both sides by filling with an electrolyte. Subsequently, a drop of lipid solution is applied directly to the surface of the aqueous electrolyte phase in one of the compartments and then waited until most of the solvent has evaporated. Thereafter, the water level is lowered by removing electrolyte volume below the aperture and raised again. As a result of the hydrophobic / hydrophilic interactions, a lipid bilayer forms at the opening. However, a direct transfer of this method to planar substrates, as they appear to be essential for automation, only partially possible. Handling is particularly difficult since the entire experimental setup must be completely stored in aqueous solution after the lipid membrane has been produced.

The authors of the article Malmstadt, N .; Nash, MA; Purneil, RF; Schmidt, JJ, Nano Lett, 2006 6, (9), 1961-1965, utilize microstructured channels in poly (dimethylsiloxane), PDMS to create a lipid membrane. In this approach, the permeability of PDMS to various solvents is exploited to continuously shrink, by diffusion and evaporation, a volume of lipid solution introduced into a microchannel, thereby creating a lipid membrane between two aqueous phases. In the approach of Suzuki, H .; Tabata, KV; Noji, H .; Takeuchi, S., Langmuir, 2006 22, (4), 1937-1942, and in particular by the authors of the article Sandison, ME; Zagnoni, M .; Abu-Hantash, M .; Morgan, H., J Micromech Microeng, 2007 17, (7), pp. 189-196, developed so-called air-exposure technique initially both sides of a comparatively large aperture (> 100 μηι) by microfluidic feeds or Zupipettieren with electrolyte solution wetted. Subsequently, a drop of lipid solution is deposited analogously to the coating technique at the opening or fed through a microfluidic channel. The aqueous phase is aspirated on one side, which allows a defined evaporation of the solvent in air. Redispersing / supplying a water drop to the aperture preserves the membrane.

However, all these known approaches have in common that the lipid bilayers can only be produced by interactions at the air phase or complicated (micro) fluidic accesses from both sides, preferably with macroscopic pumps. In addition, the conventional methods place very special demands on the experimental setup. Also, the effort to control and control electrophysiological experiments is significantly increased. These known methods are methodically unable to solve fundamental practical problems in terms of automation, speed and applicability in the generation of lipid bilayers. In particular, they are not suitable for producing lipid bilayers on very small, fluidically unidirectionally accessible apertures, which are only suitable for high-throughput applications.

Furthermore, WO 2009/069608 A1 discloses a method for producing a planar lipid bilayer membrane array, in which microchambers are formed by comb-shaped structures, each opening into a microchannel. By sequential introduction of buffer solution and lipid solution by means of a microsyringe lipid bilayers are formed in the boundary region between microchannel and microchamber. For automating the production of lipid bilayers, particularly in planned applications in high throughput studies, the system and method of making the bilipid layers shown in this document are far from reproducible and reliable enough. Above all, however, it is not possible in the known microstructure shown in WO 2009/069608 A1 to clearly detect the formation of a bilipid layer, ie by electrical measurement, since the necessary equipment of the chambers with electrodes is missing. Rather, according to WO 2009/069608 A1 optical Measuring method used, but do not allow a clear evidence of Bilipidschicht alone.

It is therefore an object of the present invention to provide a fast, reliable and simple and in real time controlled and automatable method for creating the lipid bilayers and the microstructure and the corresponding measuring device, especially in a system suitable for high-throughput investigations.

This object is solved by the subject matter of the independent claims. Advantageous developments of the present invention are the subject of the dependent patent claims.

The present invention is based on the finding that in experiments with a pipetting robot it has surprisingly been found that in some cases merely alternating application of lipid in alkane and electrolytic solution leads to an electrically contacted microcavity in a hydrophobic polymer layer to form a bilipid layer.

This highly surprising, at first infrequent, observation led to the consideration of whether or not self-assembly effects at interfaces could be responsible for this, as well as whether such effects could be more likely to occur if the movement of lipid and electrolyte were geometrically controlled, such as this z. In a microchannel or in the movement of a defined hanging drop,

These experiments were very successful to a high degree, with a high probability to detect a lipid bilayer with the characteristic high resistance and capacity. In addition, the existence of a lipid bilayer could be demonstrated by reconstitution of ion channels. It was applied to a small amount of lipid in a channel already filled with electrolyte over a microcavity in hydrophobic polymer. The following strategy proved successful:

In a microchannel already filled with aqueous electrolytes, at the bottom of which there are one or more microelectrode cavities also filled with the same electrolyte, a small amount of lipid solution is introduced, followed again by the same electrolyte. Due to the laminar flow lipid and water phase remain almost ideally separated. By further introducing electrolyte solution, the lipid phase in the microchannel is pushed further over the opening or the potty in the substrate. The lipid molecules of the lipid phase align according to their amphiphilic character at the interface with the water phase in accordance with their hydrophobic and hydrophilic components. It is to be assumed that a first monolipid layer is now formed as a result of this self-assembly process. Subsequently, a certain part of the electrolyte volume is withdrawn from the microchannel. Thus, the flow direction of the liquids is reversed and the drop is pushed back from its position over the potty or the opening. The inevitably formed parabolic flow profile leads, according to our current idea, to the application of a second monolipid layer to the first and thus to the formation of a lipid bilayer.

In addition to the hydrophobicity of the substrate material, an important prerequisite for the surprisingly high success rate is the choice of the properties of the lipid solvent tailored for this application. On the one hand, the dissolution of the lipid molecules must be completely preserved, i. H. the critical concentration for micelle formation (Critical Micelle Concentration, CMC) should be as high as possible; on the other hand, the solvent (LM) used for dissolving the lipids should be very poorly or not at all miscible with aqueous solutions. Preferably, hydrophobic solvents are used, e.g. As hexadecane, dodecane, decane, octane, hexane or pentane and mixtures of these substances, wherein the selection of pure substances or the mixture is adapted to the size dimensions of the cavity so that the fastest and safest formation of bilipid results.

In contrast to the disadvantages of existing solutions, the formation or existence of an interface with the air is not obligatory in this process. By dispensing with a third interface, additional, uncontrollable disturbances are eliminated. This leads to an increased yield in the successful generation of lipid bilayers. In addition, the expenditure on equipment is massively reduced compared to previous solutions, which partly require (micro) fluidic access both above and below the opening. This can overcome a significant limitation in the design of microapertures, which excludes single-sided closed structures as commonly used in microchip production. The production of a lipid bilayer with only one pump or one pipette is thus possible. Different lipid solutions can be introduced through a single feed. The decisive advantage of the method is that, for example, simple, commercial pipetting robots can be used to prepare the bilipid layers.

The inventive arrangement and process control results in the main advantage of this method: It is highly automated. In micro-technical systems, lipid bilayers can be produced in a single step over several openings in a single step. From basic academic research to pharmacological drug screening, the stable, simple, rapid, and reliable generation of a lipid bilayer, as an artificial cell model, is a key factor in a wide variety of applications. For the latter, particularly high experimental throughputs are extremely important for a general, productive evolution. Basic research users benefit from simplification of experimental runs, reduced equipment and systemic effort, and high variability in experiments with statistically unlikely events involving the study of membrane proteins or the model membrane itself. In summary, the method presented here is of increasing value to users In all electrophysiological and biophysical disciplines, the efficiency of experiments with model cell membranes is generally reduced by significantly reducing the time invested in performing them.

According to an advantageous embodiment of the present invention, the measuring system comprises at least one electronic data recording and control system with control and evaluation units, which provide the respectively provided for the movement of the lipid phase devices depending on the real-time values of DC resistance, impedance and / or Capacity control. In this way, an automatic or semi-automatic, feedback-controlled movement of the lipid phase on the aperture to and from the aperture away and an automatically controlled formation of a bilipid layer can be made possible.

For a better understanding of the present invention, this will be explained in more detail with reference to the embodiments illustrated in the following figures. The same parts are provided with the same reference numerals and the same component names. Furthermore, individual features or combinations of features from the embodiments shown and described can in themselves represent independent inventive or inventive solutions. Show it: 1 is a schematic sectional view of a microstructure during the overflow with lipid solution in a first flow direction;

Fig. 2 is a schematic sectional view of the microstructure of Fig. 1 when stopping the lipid solution over a first cavity; 3 shows a schematic sectional view of the microstructure during the overflow with lipid solution in a second flow direction;

4 shows a schematic sectional view through a microstructure with an electrode arrangement for monitoring the production of the bilipid layers;

Fig. 5 is a block diagram of a microstructure with integrated micropump; 6 shows an overview of a microtechnical production method for producing the arrangement from FIG. 4;

7 is a perspective view of a microstructure according to the invention with four

microcavities;

8 shows an example of a time diagram measured with the biological model system

alamethicin

Fig. 9 is a histogram of the measurement of Fig. 8;

10 shows an enlarged detail of the measurement results from FIG. 8.

FIG. 1 shows a microstructure 100 according to the invention during a first step of the production of lipid layers. In its simplest form, the microstructure 100 comprises a substrate 102, in which at least one microcavity 104 is formed. Hereinafter, these microcavities 104 are also referred to as pots, openings or apertures and are intended to denote unilaterally open cavities having dimensions of less than about one millimeter. These dimensions are mainly determined by the still stable size of the lipid layer spanning it freely suspended. Membrane-forming lipids are lipids that have a hydrophilic and a hydrophobic part-that is, amphiphilic-that allows them, in polar solvents such as water, to use either micelles (spherical or non-polar) depending on their nature Ag gregates of amphiphilic molecules that spontaneously assemble in a dispersion medium) or double lipid layers - whereby the hydrophilic moiety always interacts with the polar solvent. From these double lipid layers, with the exception of the membranes of Archaea, all biomembranes are constructed, which delimit the contents of a cell against the environment. In the present application, the terms bilipid layer, double lipid layer and lipid bilayer are used synonymously.

Above the microcavities 104, a microchannel 106 is arranged. The microchannel is bounded by a cover layer 108 on the side facing away from the microcavities 104 and is formed, for example, in a cover plate. According to the invention, the microchannel 106 and the microcavities 104 are first filled with an aqueous phase 110. As shown in FIG. 1, a lipid phase 112 is introduced into the aqueous phase 110. By way of example with the aid of a micropump or a syringe, the lipid phase 1 12 is directed over the first aperture 104 (toward the aperture) in the direction 114 (forming a laminar flow profile). The lipid molecules 116 arrange themselves, as shown schematically, so that their hydrophilic ends are oriented towards the aqueous phase 110, while their lipophilic ends are oriented towards the lipid phase 112. The material of the substrate 102 is either completely made of hydrophobic material or surface-coated, so that the lipid molecules a hydrophobic surface is presented. As symbolized in FIG. 2, in a next process step, the passage over the microcavity 104 stops and a lipid monolayer can form over the opening of the microcavity 104.

According to the invention, by reversing the pumping pressure, the lipid phase 1 12 is again moved in the opposite direction 1 18 from the aperture 104, forming a laminar flow profile. In this case, as shown symbolically in FIG. 3, the desired lipid bilayer forms above the water-filled microcavity 104. This, as will be clear from the later explanations, reproducible formation of Bilipidschichten arises by the directed movement of a defined volume of a lipid solution on the cavity and back away. As further shown in Fig. 4, the movement of the lipid solution volume can be fed back directly via the measured electrical parameters, in particular the ohmic resistance and the capacitance. The microstructure according to FIG. 4 comprises, in addition to the components already explained with reference to FIGS. 1, 2 and 3, at least one measuring electrode 120 as well as at least one counterelectrode 122. With the aid of this arrangement, measurements such as in the PhD thesis Baaken, Gerhard: "Entwicklung a microsystems engineering platform for parallel studies of ion channels in artificial cell membranes ", Albert-Ludwig University, Freiburg im Breisgau, November 2008, is performed.

In particular, by applying a defined, time-variable potential difference, the current flow between the measuring electrode 120 located in the cavity and the counter electrode 122 located outside the cavity 104 can be detected. The different electrical conductivity of the aqueous electrolyte phase 110 and the hydrophobic solvent 112 cause changes in the current amplitude. This can be used according to the invention for the detection of the hydrophobic solvent volume over the cavity and can be used in the case of a direct connection to an electrically controllable pump for a feedback to the pump. Furthermore, the proper functioning of the applied bilipid layer can be checked immediately.

As an electrode material for the electrodes 120 comes on the one hand, a noble metal such as gold in question. Preferably, however, in order to change the electrochemical behavior of the polarizable gold electrodes by means of a microgalvanic coating into the behavior of a non-polarizable electrode, the gold electrode can be coated so that it represents a silver-silver chloride electrode. This can be done, for example, according to the method of the above-mentioned Baak thesis. Also, the counter electrode 122 may be provided with a corresponding coating to form a silver-silver chloride electrode and thereby obtain a stable reference potential. Other possible embodiments of the electrodes include coating with platinum black or iridium oxide.

As shown schematically in FIG. 5, a corresponding pump 126 can be directly controlled with such an electrode system and an associated control and evaluation unit 124. Such a closed control loop enables a fully automated coating of such microstructures. Of course, the pump 126 need not be a micropump, but may be any other suitable shape. However, the provision of a micropump allows the microstructure with the micro cavities 104 together with the pump and optionally also together with the electronics of the control and evaluation unit 124 is produced as an integrated microsystem.

With reference to FIGS. 6 and 7, the fabrication of a microstructure having four microcavities 104 will be explained below. In a first step, a substrate, for example made of glass, is provided. Spin-coating and exposing a negative resist provides a mask for depositing the metal layers for the measurement electrodes 120. These are generated by evaporation, as shown in step IV, for example. In step V, the photoresist layer is removed so that the excess metal surfaces are thereby removed. According to the invention, the wall of the microcavities 104 consists of a SU8 photoresist. This can be produced directly via a photographic technique with the desired structures. As shown in step VIII, the required metallization for producing a silver-silver chloride electrode can now be applied by means of an electroplating technique.

Fig. 7 shows a perspective view of the resulting chip, by attaching a cover layer, for. B. in the form of another glass structure, to a closed system. In this cover layer, which is not shown separately here, the channel structures are also made, for example, each connect two of the apertures 104 according to the arrangement of Figures 1 to 3 with each other.

With the aid of the model protein alamethicin described in Chapter 4 of the PhD thesis Baaken, Gerhard: "Development of a Microsytronic Platform for Parallel Studies of Ion Channels in Artificial Cell Membranes", Albert-Ludwig-University, Freiburg im Breisgau, November 2008, it can be proven that Actually functionally active bilipid layers have formed above the apertures Figures 8, 9 and 10 show by way of example the results of a measurement carried out on the microstructures according to the invention.

Experiments were carried out in which lipid bilayers with incorporated A-lamethicin channels were applied and measured. As explained in detail in Baak's dissertation, alamethicin forms pores in membranes, exclusively in bilipid layers, depending on an applied transmembrane potential. Therefore, by applying a potential between the measuring electrode 120 and the counter electrode 122, a current flow through the pores in the bilipid membrane can be measured. Are alamethicin channels successfully inserted into the lipid layers spanning the pots? The measured current shows typical fluctuations that resemble a step function with different levels of conductivity. These different levels of conductivity represent pores with a different number of alamethicin monomers.

8 shows an overview of the current profile with a constant holding voltage as a function of time. FIG. 9 shows a histogram of the measured values from FIG. 8, which proves that an electrically very high-resistance layer has formed, and FIG. 10 shows an enlarged detail of the time range between 510 and 520 ms. In particular, the staircase shape of the current profile in FIG. 10 represents the characteristic curve for a functional bilipid membrane with alamethicin pores incorporated therein, in accordance with the results of the PhD thesis Baaken. This behavior rules out that only a disordered accumulation of protein is responsible for the high insulation resistance.

The measurement results of FIGS. 8 to 10 thus clearly demonstrate that the production method according to the invention produced a functional lipid double layer over the corresponding potty in the channel.

Claims

PATENT CLAIMS

1. Method for producing a bilipid layer over a microcavity that is open on one side and formed in a substrate with the following steps:

filling the microcavity and partially covering the substrate containing the microcavity with an electrolyte solution so that there is continuity between the electrolyte solution in the microcavity and the electrolyte solution on the substrate;

moving a fluid containing dissolved lipids forming a lipid phase within the aqueous electrolyte solution covering the substrate in a first direction toward the microcavity;

adjusting and monitoring the position of the lipid phase on the microcavity by detecting an increased DC resistance or impedance between a counter electrode connected to the outside of the cavity and a measuring electrode arranged inside the microcavity;

moving the lipid phase in a second direction away from the microcavity;

Monitoring the formation of the bilipid layer over the microcavity by detecting a DC resistance, an impedance and / or a capacitance between a counter electrode connected to the fluid and a measuring electrode arranged inside the microcavity.

2. The method according to claim 1, wherein the movement of the lipid phase takes place in a liquid stream.

3. The method of claim 2, wherein the first and second directions are opposite and the flow is laminar.

4. The method according to claim 1, wherein the moving of the lipid phase is carried out by moving a hanging drop of this fluid.

5. The method of claim 4, wherein the hanging drop adheres to a movable pipette or to a planar substrate adapted to move a drop.

6. The method according to any one of the preceding claims, wherein a defined time-varying electrical potential difference is applied to detect the impedance between the measuring electrode and the counter electrode and a change in an amplitude of an electrical current between the electrodes is monitored.

7. The method according to claim 6, wherein the time-varying voltage difference has the form of square-wave pulses with amplitudes between 1 and 100 mV, preferably between 1 and 30 mV, and a duration of 5 ms to 500 ms, or wherein the time-varying voltage difference has the form of ramps with a peak amplitude between 1 and 100 mV, preferably between 1 and 30 mV, and a duration of 5 ms to 500 ms, or wherein the time-varying voltage difference has the shape of a sinusoid with a peak-to-peak amplitude of 1 to 500 mV and a frequency of 0.1 Hz to 1 MHz, preferably 1 Hz to 20 kHz.

8. The method according to any one of the preceding claims, wherein the fluid comprises a hydrophobic solvent.

9. The method according to claim 9, wherein the hydrophobic solvent comprises hexadecane, dodecane, decane, octane, hexane or pentane as well as mixtures of these substances, the selection of the pure substances or the mixture being adapted to the size dimensions of the cavity so that the fastest possible and safe formation of the bilipid layer results.

10. The method according to any one of the preceding claims, wherein the ohmic resistance and the capacitance between the measuring electrode and the counter electrode are monitored.

11. Microstructure for examining lipid bilayers, wherein the microstructure (100) has a substrate (102) in which at least one microcavity (104) open on one side is formed, with at least one measuring electrode (120) inside the microcavity (104). is arranged and wherein the at least one microcavity (104) is connected to a fluid channel (106) in such a way that the microcavity can be flowed over by a laminar fluid flow with at least two different flow directions.

12. Microstructure according to claim 1 1, wherein the measuring electrode forms the bottom of the cavity.

13. Microstructure according to claim 1 1 or 12, wherein at least one counter electrode (122) is arranged outside the microcavity in order to electrically contact the fluid which covers the substrate.

1 . Microstructure according to claim 13, wherein the at least one counter electrode is located in or electrically connected to a fluid channel (106).

15. Microstructure according to one of claims 1 1 to 14, wherein a plurality of microcavities (104) arranged in an array are provided in the substrate (102).

16. Microstructure according to one of claims 1 1 to 15, wherein each of the plurality of microcavities (104) has at least one electrically separately contactable measuring electrode (120).

17. Microstructure according to one of claims 1 1 to 16, further comprising a micro pump (126) for bidirectionally conveying the fluid.

18. Microstructure according to claim 17, wherein the micropump (126) is integrated in the substrate (102).

19. Microstructure according to one of claims 1 1 to 18, wherein the substrate (102) is made from a hydrophobic material or is provided with a hydrophobic surface coating.

20. Microstructure according to claim 19, wherein the substrate (102) is formed by a photostructurable epoxy resin on a glass support.

21. Microstructure according to claim 20, wherein the at least one measuring electrode (120) is formed by a conductor track structure on the glass carrier.

22. Microstructure according to one of claims 11 to 21, wherein the at least one measuring electrode (120) is a silver/silver chloride electrode.

23. Microstructure according to one of claims 1 1 to 22, wherein the at least one microcavity has a largest diameter of 1 μm to 300 μm, preferably 2 μm to 100 μιτη, and an aspect ratio between 0.1 and 100, preferably between 0.3 and 10.

24. Measuring arrangement containing a microstructure according to one of claims 1 1 to 23, wherein the measuring electrodes and at least one counter electrode are connected to an amplifier suitable for electrical resistance, impedance and / or capacitance measurements, preferably a potentiostat, voltage clamp or patch -Clamp amplifier, are connected.

25. Measuring arrangement containing a microstructure for examining lipid bilayers with a substrate (102) in which at least one microcavity (104) open on one side is formed, at least one measuring electrode (120) being arranged inside the microcavity (104) and wherein the measuring electrodes and at least one counter electrode are connected to an amplifier suitable for electrical resistance, impedance and / or capacitance measurements, preferably a potentiostat, voltage clamp or patch clamp amplifier, the microstructure being in an upright or inverted microscope is integrated for optical observation and analysis of the bilipid layer.

26. Measuring arrangement containing a microstructure for examining lipid bilayers with a substrate (102) in which at least one microcavity (104) open on one side is formed, at least one measuring electrode (120) being arranged inside the microcavity (104) and wherein the Measuring electrodes and at least one counter electrode are connected to an amplifier suitable for electrical resistance, impedance and / or capacity measurements, preferably a potentiostat, voltage clamp or patch clamp amplifier, the microstructure for moving the lipid phase in the fluid flow or in The shape of a hanging drop is integrated into an automatic pipetting machine.

27. Measuring arrangement containing a microstructure for examining lipid bilayers with a substrate (102) in which at least one microcavity (104) open on one side is formed, at least one measuring electrode (120) being arranged inside the microcavity (104) and wherein the Measuring electrodes and at least one counter electrode are connected to an amplifier suitable for electrical resistance, impedance and/or capacity measurements, preferably a potentiostat, voltage clamp or patch clamp amplifier, whereby the microstructure is connected to a pump for generating fluid flows in the microstructure.

28. Measuring arrangement containing a microstructure for examining lipid bilayers with a substrate (102) in which at least one microcavity (104) open on one side is formed, at least one measuring electrode (120) being arranged inside the microcavity (104), wherein the measuring electrodes and at least one counter electrode are connected to an amplifier suitable for electrical resistance, impedance and / or capacity measurements, preferably a potentiostat, voltage clamp or patch clamp amplifier, wherein an electronic data recording and control system with control - and evaluation units are provided to control the devices provided for the movement of the lipid phase depending on values of direct current resistance, impedance and / or capacity recorded in real time.

29. Measuring arrangement according to one of claims 24 to 28, wherein in the bilipid layer there is at least one membrane protein that spans the membrane or is associated with it, so that the bilipid layer has at least one ionic conductivity and / or at least one other with electrical or biophysical properties that can be verified using optical methods.