WO2001034764A2

WO2001034764A2 - Apparatus and methods for positioning and analyzing biological membranous objects

Info

Publication number: WO2001034764A2
Application number: PCT/IB2000/001625
Authority: WO
Inventors: Christian Schmidt
Original assignee: Cytion S.A.
Priority date: 1999-11-08
Filing date: 2000-11-08
Publication date: 2001-05-17
Also published as: WO2001034764A3; AU1047401A

Abstract

Apparatus for positioning a cell or other biological membranous object within a confined compatible fluid compartment of fixed geometry has a surface that has a first zone repulsive to the fluid enclosing a second zone that is attractive to the fluid. The first zone controls the geometry and position of the fluid compartment on the second zone. The second zone has an area of less than about 500 νm2. A method for positioning a membranous object in the second zone on the carrier is also provided. The membranous object is first immersed into the liquid in a volume of no greater than about 100 nl. The liquid having the object immersed therein is loaded onto the surface so that the fluid is restricted to the second zone and the object comes into contact with the second zone.

Description

APPARATUS AND METHODS FOR POSITIONING AND ANALYZING BIOLOGICAL MEMBRANOUS OBJECTS

Field of the Invention This invention relates to apparatus and methods for analyzing cells, and in particular to apparatus and methods for positioning a cell or other membranous object and analyzing the object.

Background of the Invention

All biological cells have cell membranes. Cell membranes are important to the functions of a cell, including absorbing nutrients, secreting toxic waste, controlling cell volume, and communicating with the outside environment. Cell membranes contain many different classes of proteins involved in these and other functions. Important classes of proteins include, for example, ion channel proteins that control the ionic flux across the membrane.

Analysis of electric currents controlled by ion channel proteins is referred to as "functional analysis" or "functional screening." Functional screening is used in medical diagnostics, biosensorics, and drug discovery. Functional screening of ion channel proteins, analysis of the fluorescence of the ion channel proteins, and analysis of substances related to these proteins is necessary for acquiring a basic understanding of cellular processes in biological systems and for the practice of medicine.

One method for providing functional screening by current analysis is described in PCT published patent application no. WO 1998 IB 0 001 150. Other methods for electrophysiological analysis of ion channel proteins and other biological membranous objects are described in the first edition of Single-Channel Recording, edited by B. Sakmann and E. Neher and published in 1983 by Plenum Publishing Corporation. However, the apparatus and methods described in these publications have drawbacks and disadvantages that limit their application. It would be desirable to provide different and improved apparatus and methods for positioning and analyzing cells and other biological membranous objects.

Summary of the Invention

The invention provides an apparatus for precisely micropositioning a biological membranous object, including, for example, biological cells, liposomes, or portions thereof. The apparatus has a surface having first and second distinct zones. The first zone is repulsive against a liquid that is compatible with the objects to be positioned and in which the objects are immersed. The second zone is attractive to the liquid. The first zone normally circumscribes the second zone and is immediately adjacent to the second zone. The first zone controls the shape and location of the liquid in contact with the second zone. The second zone is small and has an area of less than about 500 μm². More typically, the area is less than about 200 μm . Somewhat more typically, the area of the second zone is less than about 100 μm .

This invention also provides a method for precisely positioning a biological membranous object on the apparatus of the invention. In the practice of the method, the membranous object is first suspended in a small volume of a suitable compatible liquid. For example, a physiological buffer will typically be used. Generally, the volume of buffer is from about 5 to 200 nanoliters ("nl"). More typically, the buffer volume is from about 10 to 100 nl.

The compatible liquid, having the biological membranous objects of interest suspended therein, is loaded onto the apparatus of the invention. The liquid is restricted to the second zone on the apparatus and the object comes into contact with and adheres to the second zone within a short time because of the distinct repulsive properties of the first zone and attractive properties of the second zone. Thus, the membranous objects are positioned on the apparatus in contact with the second zone. The apparatus and the method of the invention for positioning a membranous object are useful in the electrical and optical analyses of cells, cell membranes, membrane proteins, lipid bilayers, liposomes, and the like. The invention can be used, for example, to analyze biological cells or components by various techniques, including voltage clamping, voltage sensing, and impedance spectroscopy techniques. The method of this invention is simple and does not require the degree of skill normally required by conventional electrophysiological methods.

The foregoing and other advantages and features of the invention, and the manner in which the same are accomplished, will become more readily apparent upon consideration of the following detailed description of the invention, taken in conjunction with the accompanying drawings and examples, which illustrate preferred and exemplary embodiments.

Brief Description of Drawings Figure 1 illustrates one embodiment of the apparatus of this invention in a schematic sectional front planar view;

Figure 2 illustrates another embodiment of the apparatus of this invention for obtaining electrical measurements on cells and liposomes or the like in a schematic sectional front planar view; and

Figure 3 is a voltage-clamp recording taken from membranous objects positioned on a specific embodiment of the apparatus illustrated in Figure 2 and is a record of current at three different times taken over the time period indicated and at the voltage indicated.

Detailed Description of the Invention Figure 1 is a schematic sectional diagram illustrating a front planar view of one embodiment of an apparatus 10 of the invention. Apparatus 10 comprises a body 12, the upper surface of which is covered by a hydrophobic material 14. An aqueous carrier fluid 16 for biological membranous objects contacts the body 12. Direct contact between the body and the fluid is possible only at the contact zone 17, which is not covered by the hydrophobic material. Hydrophobic material 14 constitutes a first zone, which is repulsive to the carrier fluid. The contact zone which is characterized by the absence of hydrophobic material, constitutes a second zone, which is attractive to the carrier fluid. Repulsion between the hydrophobic surface coat 14 of the first zone and the aqueous carrier fluid 16 creates a freestanding fluid compartment in contact with the contact zone 17 and defined by surface 18 and maintained by the surface tension that is characteristic of the fluid. Cells, liposomes, or other biological membranous objects 20 are contained within carrier fluid 16 within compartment 18. The objects drift into the contact or second zone 17 by sedimentation due to the shape and location of the fluid compartment above the second zone.

Figure 2 is a schematic sectional front planar view of an apparatus 22 of the invention having two separate fluid compartments, 24 and 26, which can be the same or different fluids. The arrangement illustrated in Figure 2 is especially useful for taking electrical measurements of biological objects, including cells or liposomes.

The carrier fluid 28 is contained in compartment 24. The body of the apparatus is a silicon chip that comprises a bulk silicon body 30 having an adherent insulating diaphragm 32 thereon that separates the two fluid compartments. An aperture in diaphragm 32 is shown at 34 between the two fluid compartments and provides electrical communication between the two compartments. The carrier fluid 28 in compartment 24 is restricted by a hydrophobic coat 36 over the insulating diaphragm 32 that is repulsive to the carrier fluid and defines the first zone. The fluid 38 in compartment 26 is also restricted by hydrophobic coat 36, further defining the first repulsive zone. Similar to the embodiment illustrated in Figure 1, repulsion between the hydrophobic surface coat 36 and the aqueous carrier fluid of compartment 24 and between the hydrophobic surface coat 36 and the fluid of fluid compartment 26 creates freestanding fluid compartments 24 and 26, respectively, maintained by the surface tension that characterizes each fluid. The contact zone for the objects of interest is that surface area 39 that is in contact with the fluid 28 in compartment 24 and includes the aperture 34 and is limited by the repulsion zone defined by surface 36.

Compartment 24 contains the biological membranous particles or objects of interest. Vesicles, cells, or other membranous particles sedimenting in the contact zone of compartment 24 cover the aperture 34 to physically separate the two fluid compartments 24 and 26. The membranous object covering the aperture can be referred to as a "patch." Current passing between the two compartments flows through the portion of a biological membrane covering the aperture and defines the "membrane current." The membrane current can be used for analyzing membrane proteins, including ion channel proteins.

The potential difference driving the current is applied by electrodes 42 and 44 located opposite each other in the fluid compartments 24 and 26, respectively. If desired, an insulating layer 40 can be placed on the silicon body opposite the insulating diaphragm 32 to reduce or preclude capacitive noise and other artifacts of electrical measurement.

The apparatus of this invention can be used to carry or position any biological cells, artificial simulations of biological cells, liposomes, or portions thereof, including, for example, portions of cell membranes, protoplasts, and cell organelles. Cell organelles include, for example, mitocondria, chloroplasts, lipid bilayers, lipid micelles, and the like. The apparatus of this invention is useful for positioning many other cell-like materials including viral particles, mycoplasms, macromolecular complexes, and the like. The apparatus and method of this invention are especially useful for obtaining optical and electrical measurements on membranous objects.

The apparatus provides electrical and optical access to the cellular and liposomal membrane proteins of interest, when necessary, and also provides precise positioning of membranes on apparatus, which is important for taking optical and electrical measurements on membranes.

As illustrated in connection with Figures 1 and 2, the invention provides positioning of particles at a specific site or contact zone on a carrier by providing for adhesion of the particles only at the specific site. The space available for movement of the particles is confined to the close vicinity of the adhesion site by the geometry of the fluid compartment in which the particles are carried. The fluid compartment that contains the particles is confined to a particular geometry by the construction of the carrier and by the size of the carrier and the amount of fluid used so that the fluid stays in contact with the site. The particles are immersed in fluid that is in contact with the part of the carrier surface containing the contact zone. The particles are immersed in a fluid that is compatible with the particles and does not adversely affect the physical, chemical, and physiological state of the immersed particles. Normally, this fluid is a liquid and is a compatible aqueous solution. For example, a cell culture medium or a modified cell culture medium would be used for a biological cell. Other buffers and solutions useful for suspending, diluting, or storing the particles can typically be used. The fluid should not interfere with or adversely affect the attachment of the particle to the contact zone on the cell carrier. The fluid should not affect the analysis of the particles on the carrier, including electrical recording and optical analysis. Normally, physiological buffers, including PBS, or Hank's buffer, and the like, will be useful. In the embodiment of Figure 2, two fluids are used, one as the carrier fluid

28 and one opposite the carrier fluid 38 for electrical transmission. These fluids can be the same or different, but the fluid 38 should be selected from the same group set forth above.

Precise positioning of particles on the carrier is achieved by providing, simultaneously, 1) surface patterns on the carrier that allow adhesion between the particle and the carrier only at the contact zone, but substantially precludes adhesion of particles outside the contact zone; 2) a defined fluid compartment in which the particles are immersed at the contact zone so that the movement of the particles is restricted to the vicinity of the contact zone, which normally is less than about 100 to 500 μm into the fluid compartment, depending on the particle size, and 3) directed motion of the particles towards the contact zone and random motion of the particles within the fluid compartment.

Directed motion is due to force fields, including gravitation, electrophoresis, and dielectrophoresis. Random motion is due to Brownian motion and convection. Together, these forces enable precise positioning within the fluid compartment after the compartment has been created at the contact zone.

A surface patterning that allows adhesion between the particle and the carrier only at the contact zone and precludes adhesion to all other places can be accomplished by creating the first zone repulsive surface and restricting the adhesive contact zone which is a portion of the apparatus main body 12 (Figure 1) and 30 (Figure 2) to a total area of from about 0.1 to 500 μm . Usually the total area of the contact zone will be from about 0.5 to 200 μm . Somewhat more typically, the total contact area will be from about 1 to 100 μm².

The defined, self-contained fluid compartment at the contact zone is created by adjacent attractive forces at the contact zone and repulsive forces outside the zone acting on the fluid. These forces may have different origins, including electrical charge interactions and hydrophilic/hydrophobic interactions. Physically or chemically patterned surfaces for the body of the apparatus are mainly hydrophobic surfaces that contain small hydrophilic contact zones within them. Various carrier materials can be used in the practice of the method of the invention, including, for example, SiO , Pyrex™, and various plastics in various geometries and sizes. Electrostatic interactions between these hydrophilic zones and the particles can occur that are particularly useful. For example, negatively charged giant liposomes can be attached to a positively charged planar surface contact zone of about 1 to 100 μm² prepared from Siθ2 modified with physisorbed poly-L- lysine and surrounded by a hydrophobic material, including, for example, Teflon^® or Si₃N₄. Other hydrophobic materials, including the silanes in the case of SiO₂ and Pyrex™ carrier surfaces, and photosensitive coats allow the definition of contact zones by assembly and by photolithographic techniques, respectively. The specific geometries and combinations of materials useful in the practice of the invention are too numerous to mention and are believed to be well within the scope of the abilities of the skilled artisan once apprised of the examples of this disclosure.

Contact zones can be produced by partial activation of hydrophobic carrier surfaces. Surfaces can be activated by applying an oxygen plasma, high energy radiation, including UV radiation, or reactive chemical compounds, including Cl and Br_2> to the surface area intended to be the contact zone. For example, an oxygen plasma can be applied to a PDMS carrier whose surface is covered by a mask that allows plasma access only at the contact zone. The same technique is also applicable to SiO and similar carrier surfaces, which can be covered with, for example, silanes or polyaminoacids. If necessary, the activated surface can be further modified by physisorption or covalent attachment of functional groups that promote the binding of selected cells or vesicles, as is described further hereinbelow. For example, the contact zone can be made selective for certain types of cells or liposomes. The contact zone can be modified for specific interactions with the surface of the cells or liposomes. The contact zone can be coated with materials having binding affinity to the cell surface or liposome surface. Suitable materials can include, but are not limited to, biotin, avidin, laminin and the laminin receptor, integrin and integrin receptors, and the like. In general, interactions between a membrane receptor and a suitable ligand, or substrate in general, can be used for affinity binding. Thus, only cells or their organelles with specific properties are permanently positioned. The contact zone can also be treated with antibodies and antigens in a similar manner so that certain objects are attached to the contact zone. For example, the second zone, which is the adhesion or contact zone, can be coated with antibodies that attach to certain specific molecules or structures on the cells or liposomes that it is desirable to position on the carrier. The invention provides small fluid volumes that restrict the possible movement of the particles to allow positioning. For small fluid volumes of less than about 10 to 100 nl, the time required for cells or liposomes to enter the attractive range of the attractive contact zones for positioning becomes very small, and usually less then 5 minutes. The smaller the fluid volume in which the particles are suspended, the higher the chance to touch the contact zone or to enter the attractive range of this zone where charge interactions are operative. Also, the smaller the compartment size, the shorter the time required for positioning. For these reasons, the sample volumes in which the particles of interest are suspended should be as small as possible during the positioning process. Normally, the volume can be in the range from about 1 nanoliter to 500 nanoliters. Somewhat typically, the volume is from about 5 nl to 200 nl. Even more typically, the volume is from about 10 nl to 100 nl.

Other factors, including the rate of evaporation and the minimal buffer volume required for a specific cell to survive, may impose a compromise on the actual volume used. However, a very small fluid volume can be used for fast attachment of the particle to the specified contact zone and the fluid volume can be increased immediately thereafter. Using a two-step process in this manner may allow small volumes for positioning and short positioning intervals without impairing biological particles.

Another way of using very small volumes without the danger of evaporation of significant volume percentages is the employment of essentially inert and hydrophobic fluids overlaying the actual buffer volume. These fluids should be selected for a low dielectric constant and dissipation factor for sensitive electrical measurements. These fluids, an example of which is an alkane, including, for example, decane, can be added after the buffer fluid or before if adequate means are provided. A pipette can be used to dispense the buffer directly at the interface of the cell carrier surface and the inert fluid. Fluid injection can also be accomplished by ink jet technology and is believed to be within the purview of the skilled artisan once apprised of this disclosure.

After positioning onto a carrier of defined geometry, the exact location of the membranous object attached to the carrier is the place of the contact zone and is thus predetermined. The predetermined location of the membranous object provides the basis for constructing optical apparatus, and, in particular, confocal optical apparatus, for observation of the membranous objects. For example, cell membranes can be observed with high numerical aperture objectives after positioning the cells on a thin planar and transparent carrier, including glass. Examples of optical methods suited for such a use include fluorescence correlation spectroscopy (FCS), single molecule detection, and simple fluorescence observation of the cytosol. The latter does not require high numerical aperture objectives because of the three-dimensional size of the cytosol and the freedom to place the confocal spot within the cytosol. The cytosol can be loaded with fluorescent dyes that serve as probes for various cellular parameters, including free calcium concentration and membrane potential.

Optical and electrical analysis methods can be combined in one single setup enabling electrical and optical measurements on particles to be performed simultaneously on membranous objects positioned in accordance with the invention. Thus, new insights can be obtained into the molecular behavior of single molecules. As an example, the embodiment of Figure 2 may include a fully flat insulating diaphragm 32 onto which the membranous object of interest is positioned. The diaphragm may also serve as the carrier described in connection with confocal optical techniques and then subsequently provide for the concurrent optical and electrical analysis of the patch, or membranous object, covering the diaphragm aperture. The size of the various set-ups and the combined opto-electrical set-up described above can be very small, less then 1 mm x 1 mm x 1 mm. The small size can be achieved due to the small size of the fluid compartments and carrier structures. This miniaturization makes it possible to build miniaturized biosensors and to integrate multiple set-ups into one single device, or to use one single carrier for multiple set-ups, as required for HTS.

Figure 3 shows a voltage-clamp recording for a more specific embodiment of the cell carrier as illustrated in Figure 2. The carrier can be fully or partly insulating and separate two fluid compartments as illustrated in and described above in connection with Figure 2. The fluid compartments can be electrically connected by a small fluid passage through the carrier such as aperture 34 in the contact zone. The apparatus can be fully covered by positioning of a membranous object over the aperture in the contact zone. After positioning and electrically tight binding of the object over the passage, a voltage is applied between the two compartments via electrodes and the resulting current is analyzed. Electrically tight binding means that complete coverage of the aperture is achieved. The current that results thus represents the actual membrane current mediated by transmembrane proteins and the leak currents flowing between the membrane and carrier body. A tight bond between surface and particle, referred to as a tight seal, reduces the leak current and allows sensitive current measurements. Tight seals of greater than 10⁹ Ohm can be achieved that allow low noise analysis of single ion channel proteins.

The membrane currents can be measured as follows for an electrically insulating carrier that separates the two fluid compartments and has a small aperture of diameter of from about 0.1 to 10 μm within the contact area that connects the fluid compartments. A voltage is applied across the liposome or cell or their respective membranes that covers the aperture by immersing redox electrodes into the fluid compartments. The current, which flows between the two conductive fluid compartments, is mainly determined by the resistance of the membrane patch covering the aperture, or by rupturing the patch by the resistance of the entire membrane.

A micromachined silicon Si/Si₃N₄/SiO chip is used as the carrier body 30 in the embodiment of Figure 2 from which the data in Figure 3 was obtained. The silicon body 30 is covered by an insulating diaphragm 32 comprising Si₃N₄/SiO₂. This diaphragm has been made accessible from the silicon body side by silicon etching. The access from the opposite side is restricted to an adhesive or contact zone that surrounds an aperture 34 located within the freestanding part of the insulating diaphragm 32. Placement of cells and vesicles over the diaphragm aperture allows electrical recordings as described below in connection with Figure 3.

The contact zone used in the embodiment of Figure 2 from which the Figure 3 data was obtained had a diameter of 15 μm and the diameter of the aperture was 3 μm. The contact zone was formed of SiO covered by physisorped Poly-L-Lysine. Physisorption was accomplished by 12 hours immersion of the carrier body in a solution of 0.1% by weight of Poly-L-Lysine that was provided by Sigma Diagn. Inc. under their stock no. P8920. After addition of 85 mM KC1 at a pH of 7.2 as a buffer on both sides of the carrier fluid and insertion of Ag/AgCl redox electrodes, unilamellar vesicles having a density 10⁶ to 10⁷ vesicles/ml and consisting of 70% asolectin, 25% l-palmitoyl-2-oleoyl-5«-glycero-3-[phospho-rac- (1-glycerol)] (POPG), and 5% cholesterol by weight suspended in H O containing 200 mM Sorbitol, were added to the upper compartment 24, as shown in Figure 2, to a total volume of about 200 nl. Current traces are shown in Figure 3 before the addition of vesicles at time

0, again after about 30 sec after addition of the vesicles, and then at about 4 minutes after the addition of alamethicin, which is a pore forming peptide that produces typical current modulations at the applied voltage V. All traces of current were referenced to the 0 pA base line, which is shown in Figure 3 as a dashed line. To avoid electrophoretic positioning and other undesirable artifacts of current measurement, a voltage clamp was turned off during positioning and positive voltages were not applied until afterwards. This action counteracted electrophoretic positioning during current measurement.

The redox electrodes used for voltage application and current recordings can be directly attached to the carrier. Those electrodes will usually be underneath the hydrophobic surface coat and directly in contact with the buffer interfacing the contact zone. Electrodes of Ag or Pd can be sputtered or evaporated directly onto the carrier. Conductive silver inks can be used to print electrodes onto the carrier.

Normally, it is necessary to chlorinate electrodes by immersion of the entire carrier into a Cl₂ atmosphere when working with silver. All of these methods are suited for bulk processing of large carrier quantities or carriers containing multiple contact and recording sites.

Undesirable artifacts caused by conductive or semiconductive carrier body materials, including silicon, can be reduced or eliminated by adding an insulating layer 40 to the carrier body before recording. The insulating layer reduces capacitive artifacts, including capacitive noise, that arises when a large area of the diaphragm is located between the fluid compartment and the carrier body due to an increase in capacitance C. Insulation is of importance for all types of electrical measurements and recording set-ups based on chips used for the carrier body that contain semiconductive or conductive material. Insulation is particularly useful for set-ups having large areas of a thin insulating diaphragm directly sandwiched between the semiconductive carrier body and the fluid compartment as in PCT patent application WO 1998 IB 0 001 150.

The method for positioning the membranous objects on the carrier and the apparatus described can also be combined with other types of electrical recording apparatus than the voltage clamp, including voltage sensing devices and impedance spectroscopy techniques.

The partially or fully conductive contact zone of an insulating carrier can be used as an electrode and connected to a voltage sensing device, a voltage follower.

The contacting fluid volume can be connected to ground by an electrode. The skilled artisan will recognize that the arrangement could be reversed with the carrier connected to ground by an electrode and the contacting fluid connected to a voltage follower. A cell placed onto the contact zone thus provides for recording changes in the extracellular potential. These changes can be used for analyzing cells under various environmental conditions. For such a set-up, it is important to isolate the contact zone electrode sufficiently against the extracellular fluid, which is the fluid in the fluid compartment. Standard analysis procedures used in impedance spectroscopy in connection with current-voltage and phase-shift relations are suited for the analysis of the membrane properties of cell and lipid bilayers. Applying an alternating voltage between the electrodes provides for analysis of the membrane attached to the contact zone electrode. The invention has been described in particular exemplified embodiments.

However, the foregoing description is not intended to limit the invention to the exemplified embodiments, and the skilled artisan should recognize that variations can be made within the scope and spirit of the invention as described in the foregoing specification. The invention includes all alternatives, modifications, and equivalents that may be included within the true spirit and scope of the invention as defined by the appended claims.

Claims

WHAT IS CLAIMED IS:

1. Apparatus for positioning a biological membranous object contained within a fluid that is compatible with the object, said apparatus comprising a surface having a first zone that is repulsive against the fluid and a distinct second zone adjacent the first zone that contacts the fluid, wherein said second zone has an area of less than about 500 μm² , and wherein said first zone controls the location of the fluid in contact with said second zone, whereby biological membranous objects in the fluid are positioned in contact with said second zone.

2. The apparatus of Claim 1, wherein the fluid is an aqueous liquid, then said first zone is hydrophobic and said second zone is at least partially hydrophilic.

3. The apparatus of Claim 2, wherein said fluid is a cell culture medium or a physiological buffer.

4. The apparatus of Claim 2, where the aqueous liquid is overlaid by a hydrophobic liquid that contacts said first zone.

5. The apparatus of Claim 1, wherein said first zone is selected from the group consisting of a hydrophobic plastic material, silane, and silicon nitride.

6. The apparatus of Claim 5, wherein said plastic material is PTFE or PDMS.

7. The apparatus of Claim 1, wherein said second zone has been made by a spatially selective surface treatment of a homogeneous surface so that said surface attracts the fluid.

8. The apparatus of Claim 7, where the selective surface treatment comprises a treatment medium selected from a group consisting of an oxygen plasma, UV-radiation, and reactive chemicals.

9. The apparatus of Claim 1, wherein said second zone has a surface of pure or doped SiO₂.

10. The apparatus of Claim 1, wherein said second zone is coated with a charged material.

11. The apparatus of Claim 10, wherein the charged material is selected from the group consisting of poly-L-lysine, a charged silane, and a lipid.

12. The apparatus of Claim 1, wherein said second zone is coated with a molecule capable of binding to a cell surface.

13. The apparatus of Claim 12, wherein said molecule is selected from the group consisting of lamin, biotin, avidin, integrin, antibodies, and compounds that can act as agonist or antagonist to cell membrane receptors.

14. Apparatus for positioning a biological membranous object in a fluid that is compatible with the object, said apparatus comprising a body having a surface applied thereto that is repulsive against the fluid, said surface defining a contact zone on a portion of the body not covered by said surface for attracting the fluid, said contact zone having a diameter of from 0.5 to 200 μm, and wherein a fluid volume of from about 1 to 500 nl is positioned over and covers said contact zone and the membranous objects in the fluid are in contact with said contact zone.

15. The apparatus of Claim 14 wherein said contact zone further defines an aperture and the biological membranous objects cover said aperture.

16. Apparatus for positioning a biological membranous object in a liquid that is compatible with the objects for electrical analysis of said objects, said apparatus comprising a body for separating two compartments of liquid on opposite sides thereof; a dielectric surface applied to one side of said body for separating the two liquid components and having an aperture therein providing electrical communication between the compartments, a surface covering a portion of the dielectric surface that is repulsive to the liquid and confines the liquid to a zone of contact with the dielectric surface that is defined by the repulsive surface, whereby membranous objects of interest cover said aperture, and a pair of electrodes, one in each fluid compartment, for application of voltage to cause a current to travel through the aperture and membranes covering the aperture, whereby electrical properties of biological membranous objects located over said aperture are determined.

17. The apparatus of Claim 14, where an electrode is directly attached to the carrier body surface that is in contact with the fluid.

18. The apparatus of Claim 17, wherein said electrodes are a redox electrodes.

19. The apparatus of Claim 18, wherein said aperture is filled with the fluid.

20. A method for positioning a biological membranous object, comprising: providing a body comprising a surface having a first zone defining a second zone, the first zone being repulsive against a fluid and the second zone being attractive to the fluid, wherein the second zone has an area of less than about 500 μm²; immersing an object in no greater than about 500 nl of the fluid; and loading the fluid having the object immersed therein onto the surface such that the fluid is restricted to the second zone and the object comes into contact with the second zone.

21. The method of Claim 19, wherein the second zone comprises materials capable of interacting with the object.

22. The method of Claim 21 , wherein said second zone interacts with the object through electrical charges.

23. The method of Claim 21 , wherein the second zone interacts with the object through affinity binding.

24. The method of Claim 21 , wherein the materials are selected from the group consisting of biotin, avidin, integrin, laminin, antibodies, polylysine, and substances acting as agonists or antagonists to membrane receptors.

25. The method of Claim 20, wherein the fluid is a cell culture medium or an aqueous cell buffer.

26. The method of Claim 20, wherein the membranous objects are selected from the group consisting of animal cells, plant cells, bacteria cells, fungal cells, liposomes, or a portion thereof.

27. The method of Claim 20, wherein the membranous object is a lipid bilayer.

28. The method of Claim 20, further comprising adding additional fluid to the contact zone upon the positioning of the membranous particle on the second zone, up to a maximum of about 500 nl.

29. The method of Claim 20, wherein the second zone has an aperture having an area of from about 0.01 to about 50 μm .

30. A method for studying a membranous object comprising: loading the membranous particle onto the apparatus of Claim 1 ; and analyzing the membranous particle on the apparatus.

31. The method of Claim 30, wherein said step of analyzing the membranous object comprises electrical recording.

32. The method of Claim 31 , wherein the electrical recording is a voltage clamp recording performed by applying a voltage difference across the positioned membranous object.

33. The method of Claim 32, wherein the electrical recording is obtained from an impedance-spectroscopic measurements.

34. The method of Claim 30, wherein the step of analyzing the membranous object comprises optical analysis of said membranous object.