JP2004528539A - Apparatus and methods for high throughput patch clamp assays - Google Patents

Abstract

An apparatus for measuring the electrophysiological properties of cell membranes of individual cells includes a plate with at least one opening. The opening is surrounded by a surface, which is modified, for example, by heat treatment to facilitate the formation of a giga seal. A chamber is adjacent to the plate. The chamber is in fluid communication with at least one opening and is adapted to hold an electrically conductive solution. The plate further includes a first electrode located in the chamber and a second electrode located adjacent to the plate.

Description

【Technical field】
[0001]
The present invention relates generally to the electrophysiological evaluation of biological materials. More specifically, the present invention relates to devices and techniques for measuring and evaluating electrophysiological properties associated with ion channels in cell membranes. The invention further relates to a technique for forming a gigaseal between the cell membrane and the surface of the patch clamp probe to facilitate high-throughput measurement of electrophysiological properties.
[Background Art]
[0002]
Ion channels control the flow of ions into and out of cells. Ion channels typically consist of proteins or aggregates of proteins and are embedded in lipid bilayers that make up cell membranes. The movement of ions through the cell membrane through the ion channel creates an ionic current that causes a weak but measurable current.
[0003]
The patch clamp method allows measurement of the ion current passing through the ion channel. Patch clamp techniques are described, for example, in PCT International Publication Nos. WO 96/13721 and WO 99/66329, which are incorporated herein by reference in their entirety. Briefly, the patch clamp method creates a tight seal between the membrane and the recording probe, resulting in a background ion current due to "leakage" between the cell membrane and the recording probe. Take advantage of the ability of cell membranes to minimize In today's patch clamp technique, a micropipette tip is engaged with the membrane to form a seal. Such seals, known as "giga seals" and having high resistance, facilitate accurate measurement of weak ionic currents flowing through ion channels in cell membranes.
[0004]
Many ion channels have "gates" that open in response to external stimuli. External stimuli include electrical potentials, mechanical or tactile stimuli, and signaling molecules. Signaling molecules are essentially chemical stimuli, and a class of ion channel gates that respond to chemical stimuli is known in the art as ligand-gated ion channels. Ligand-gated ion channels respond to both naturally occurring signaling molecules and synthetic molecular signals such as drugs. Examples of signaling molecules for ligand-gated ion channels include acetylcholine and glycine (neurotransmitters), cyclic AMP, inositol 1,4,5 triphosphate (IP_Three), And ATP (in a cell). The development of efficacious drugs for the treatment and treatment of patients with diseases and disorders related to ion channels has been confirmed by patch clamp assays.
[0005]
In an existing embodiment, the patch clamp method is a low-throughput assay for drug candidates. The main obstacle concerns the formation of a gigaseal between the membrane and the tip of the pipette. Today's techniques for forming giga seals are laborious and require special proficiency and equipment. At present, skilled electrophysiologists can only screen about 5-20 compounds per day using existing patch clamp techniques. In contrast, modern drug screening (e.g., screening using 96-well plates, robotic handling, and automated data processing using non-patch-clamp techniques) requires 1000 to 10,000 species per day depending on the specific assay. (Or more) compounds can be screened.
[0006]
Other existing electrophysiological recording methods include one of two microelectrode voltage clamp methods, extracellular recordings, and "U-tube" methods. Although relatively demanding in terms of equipment and human proficiency, these techniques also do not meet today's demands for high-throughput screening.
[0007]
Other possible ion channel activity recording methods, such as optical methods of recording voltage changes across cell membranes, have higher throughput. However, these methods lack accuracy and information content for screening by the electrophysiological methods described above, and cannot provide the amount of information that can be obtained from electrophysiological records.
[0008]
Accordingly, there is a long-felt need for systems and methods for measuring and assessing the electrophysiological properties of cells and cell membranes under high-throughput conditions (eg, systems and methods that significantly increase the speed at which patch-clamp-type assays are performed).
DISCLOSURE OF THE INVENTION
[0009]
Summary of the Invention
The present invention provides various devices and methods for measuring, evaluating, recording, and analyzing the electrophysiological properties of cells and biofilms. The devices and methods of the present invention utilize a gigaseal between the cell or biofilm and the orifice.
[0010]
According to one embodiment of the present invention, an apparatus and method for an automated ion channel assay utilizing a gigaseal between a cell and an opening in a glass sheet or plate, and paralleling many drug candidates and cells at once Apparatus and methods for processing and screening are provided.
[0011]
Embodiments of the present invention
An apparatus for measuring an electrophysiological property of a cell membrane of an individual cell,
A plate having at least one opening surrounded by a surface that is modified to facilitate formation of a giga seal;
A chamber adjacent the plate in fluid communication with at least one of the openings and adapted to hold a solution;
A first electrode located in the chamber, and
A second electrode located adjacent to the plate
Equipped with
Includes the devices described above for use in measuring the electrophysiological properties of cell membranes of individual cells.
[0012]
Other embodiments of the present invention include:
An apparatus for measuring an electrophysiological property of a cell membrane of an individual cell,
A plate having at least one well with an opening made using a laser and modified by heating to receive individual cells;
A chamber adjacent the plate, in fluid communication with the opening, and adapted to hold an electrically conductive solution;
A first electrode located in the chamber,
A second electrode located in the well, and
Amplifier in electrical contact with first and second electrodes
Equipped with
Includes the above device for recording the electrophysiological properties of the cell membrane of individual cells by measuring the current flowing through the first and second electrodes.
[0013]
Another embodiment of the present invention includes a removable disk having an opening that functions as part of a well for use in measuring electrophysiological properties of a cell membrane.
[0014]
Other embodiments of the present invention include:
Providing at least one well with an opening having a modified surface for receiving cells having a cell membrane;
Depositing cells in the openings and forming a gigaseal between the cells and the wells with the modified surface; and
Recording voltage and / or current measurements to evaluate ion channels in cell membranes
And measuring a current passing through an ion channel of a cell membrane.
[0015]
Other embodiments of the present invention include:
Providing at least one well having an opening;
Placing a solution containing a plurality of cells into a well;
Applying a positive pressure to the opening; and
Applying a negative pressure to the opening to draw one of the cells into the opening to form a giga seal between the cell and the opening;
And a method of forming a giga seal.
[0016]
A technical advantage of one embodiment of the present invention is that an apparatus is provided that facilitates formation of a giga seal. Such a device improves signal detection and amplification for measurement of ionic current. Further, such devices provide the property of enhancing high-throughput screening of drug candidates. In addition, a method of assembling the apparatus and components of the disclosed invention is disclosed.
[0017]
A technical advantage of one embodiment of the present invention is that a novel method is provided for forming an electrically resistive giga seal between a cell membrane and a recording probe to facilitate measurement of ionic current through the cell membrane. It is a point. Further disclosed is a method for chemically and physically modifying the surface of the probe to be attached to a cell membrane. Surface modifications that facilitate the formation of a gigaseal include, but are not limited to, (1) heat treatment for a specific period of time, (2) covalent attachment of lipid molecules, and (3) application of an adhesive-like substance. Can be In one embodiment, such modifications are used with existing patch clamp experiments and can also be used to facilitate high throughput screening procedures.
[0018]
A technical advantage of one embodiment of the present invention is that it provides a novel method for screening ion channels in a high-throughput manner. Gigaseal can be used in such screening techniques.
[0019]
A technical advantage of one embodiment of the present invention is that the number of whole cell patches that can be assayed is increased from the current 5-20 per 8 hours to 200-2000 per day (or more). is there.
[0020]
Other objects, configurations, and technical advantages of the present invention will become more apparent from the interpretation of the detailed description herein and the accompanying drawings.
[0021]
Embodiment of the Invention
The following detailed description refers to the accompanying drawings. Other embodiments are possible and modifications can be made without departing from the spirit and scope of the invention. Accordingly, the following detailed description is not intended to limit the invention. The scope of the invention is defined by the appended claims.
[0022]
For convenience of the following description, the following terminology will be used. However, these descriptions are for illustrative purposes only. They are not intended to limit the term as described or referred to throughout the specification. Rather, these descriptions are intended to cover any further aspects and / or examples of the terms set forth and claimed herein.
[0023]
As used herein, the term "biofilm" is a cell membrane as found in whole cells (whole cells), and also includes artificial membranes such as lipid bilayers and other synthetic polymer membranes.
[0024]
The term "electrophysiological property" as used herein is a measured electrophysiological property of a cell, cell membrane or other biofilm. These properties are measured using the ionic current, voltage or magnetic field associated with the ion channels present in the cell membrane. Such measurements can be made in the presence (or absence) of endogenous factors, such as signaling molecules. Such measurements can also be made in the presence (or absence) of exogenous factors such as candidate drug molecules or test compounds in the screening conditions. Measuring electrophysiological properties also involves measuring the potential across cell membranes or the rate of ion migration through ion channels in the presence of endogenous or exogenous factors. The term electrophysiological properties may also encompass characteristics of the types of ions that pass through the ion channels present in the biofilm.
[0025]
An "electrically conductive solution" is a solution that contains ions or electrolytes. An ion is a charged atom or molecule having a positive or negative charge, such as a cation or anion. Examples of cations include, but are not limited to, sodium (Na⁺), Potassium (K⁺), Lithium (Li⁺), And other monovalent cations, calcium (Ca²⁺), Magnesium (Mg²⁺), And other divalent cations. Examples of anions include, but are not limited to, chloride ion (Cl^-), Iodide ion (I^-), And other halide ions.
[0026]
"Ion channels" are transmembrane proteins or aggregates of proteins embedded in a lipid bilayer that encompasses cell membranes. Ion channels control the flow of ions into and out of cells. Ion channels can exhibit specificity, for example, allowing only specific ions to pass through cell membranes. Furthermore, various diseases and disorders are closely associated with specific ions and their corresponding channels (eg, K-channels or Na-channels). The movement of ions through the cell membrane through the ion channel creates an ionic current that causes a weak but measurable current. Thus, the same cell or cell membrane may exhibit different electrophysiological properties in the presence or absence of different ions. For example, electrophysiological measurements are not only sensitive to parameters such as the presence and concentration of molecular signals, but also to the type of ions present. Furthermore, a voltage can be applied to induce the opening of ion channels in the cell membrane.
[0027]
The term "experimental variable" refers to factors controlled by the experimenter, such as, for example, temperature, duration of the experiment (such as the duration of current measurement), method of signal detection, and applied voltage. These factors can be altered by the experimenter from experiment to experiment, and such alterations can affect the results of a particular experiment. Other experimental variables include the presence and concentration of ions (including buffers), molecular signals, or drug candidates at the screening conditions. Other experimental variables include the type and number of cells present in the experimental system.
[0028]
"Ion current" is the flow of ions through an ion channel. Ionic current may refer to the movement of ions in the electrolyte solution. If a current flows in the electrolyte solution, the charge can be carried by the movement of both anions and cations. The solvent in the electrolyte solution is often water, but non-aqueous electrolyte solutions are known to those skilled in the art and are within the scope of the present invention.
[0029]
A "patch clamp microchamber" is a device for measuring the electrophysiological properties of a biofilm in which a gigaseal is formed in a single integrated device. In addition, in one embodiment, small signal amplification and / or processing can be performed in this single integrated device.
[0030]
A "giga seal" or "high resistance seal" is a patch clamp seal with minimal background ionic current due to "leakage" due to poor sealing. In a preferred embodiment, the giga seal is a high resistance seal that is greater than about 1 giga ohm (1 GΩ). In another embodiment, the gigaseal is a high resistance seal from about 1 gigaohm (1 GΩ) to about 100 gigaohm (100 GΩ). In another embodiment, the gigaseal is a high resistance seal from about 1 gigaohm (1 GΩ) to about 50 gigaohm (50 GΩ). In another embodiment, the gigaseal is a high resistance seal from about 1 gigaohm (1 GΩ) to about 10 gigaohm (10 GΩ). In another embodiment, the gigaseal is a high resistance seal from about 1 gigaohm (1 GΩ) to about 5 gigaohm (5 GΩ).
[0031]
A "proton" is a hydrogen atom whose sole electron has been eliminated. In aqueous solution, the protons are associated with water molecules, and should correctly be "hydronium ions" or H_ThreeO⁺Called. These terms are often confused and remain in the text disclosed, particularly in the discussion of pH. Unless otherwise noted, the terms proton and hydronium ion are used interchangeably herein.
[0032]
The devices and methods disclosed herein are useful for the discovery and evaluation of drugs or other therapeutic agents that are effective against ion channel related diseases. A “therapeutic agent” (eg, a drug or prodrug) is any compound or formulation thereof that is effective to promote prevention or treatment of a disease or condition. "Effective in promoting the prevention or treatment of a disease or condition" means that when administered in a clinically relevant manner, it improves symptoms, cures, prolongs life, improves quality of life, or has other effects. Such beneficial effects refer to cases where at least a statistically significant proportion of patients will generally be found to be positive by physicians familiar with treating a particular type of disease or condition.
[0033]
Diseases associated with ion channels and ion channel function include the cardiovascular field (eg, hypertension and cardiac arrhythmias), pain (local anesthetic), diabetes, epilepsy, anxiety, and the like. Each of these diseases or families of diseases tends to be associated with a particular ion channel. In one embodiment, the present invention provides devices and methods that facilitate high-throughput experiments for identifying and screening drug candidates for the treatment of ion channel-related diseases.
[0034]
The following detailed description refers to the accompanying drawings. Other embodiments are possible and modifications can be made without departing from the spirit and scope of the invention.
[0035]
FIG. 1 shows an apparatus for measuring electrophysiological properties according to one embodiment of the present invention. This device utilizes a dielectric barrier 2 used to separate a pair of electrodes 3 and 4. The dielectric barrier may be of any configuration or form (e.g., plate, disk or sheet) and may be incorporated within another structure, e.g., at the bottom, sides or top of a well or chamber. May be. Thus, the main function of the dielectric 2 is to separate the electrodes 3 and 4. The dielectric 2 has an opening 5 for receiving a cell 6. Various methods can be used for forming and forming the opening. For example, a small conical hole can be drilled at the bottom of each well using laser drilling techniques. In one embodiment, the diameter of the opening ranges from about 1 μm to about 5 μm, preferably 2 μm.
[0036]
One or more holes are made in the cell 6, and a current value or a voltage value is collected and recorded using the measuring device 7. In one embodiment of the present invention, the dielectric (or dielectrics) is provided with multiple openings and multiple dielectrics so that multiple cells can be evaluated in parallel in multiple wells (one cell at a time). It has electrodes.
[0037]
The dielectric barrier can be composed of a variety of different materials (eg, glass or plastic). Glass with good electrical insulation is useful for patch clamp measurements. Furthermore, it is useful to prevent substances, such as ions, that can modify the behavior of the channel from leaking out of the glass. Other important aspects of glass are good cell adhesion, high resistivity, low dielectric constant, compatibility with laser drilling techniques, uniform thickness, and good flatness.
[0038]
According to one embodiment of the present invention, borosilicate glass is used in the present invention. Borosilicate glass is chemically durable, stable to deformation up to about 700 ° C., and has excellent optical properties. Thus, the glass is suitable for use where a smooth, flat, minimal thickness sheet of glass is required. A type of borosilicate glass that can be used is Erie Scientific Company's D263 glass. Information on the glass can be found at http://www.eriesci.com/products&services/CustomGlass/D263-tech.html, which is hereby incorporated by reference in its entirety. Alternatively, other silicate glasses or other types of glass can be used.
[0039]
In another embodiment, soda lime glass can be used for the plate portion of the patch clamp microchamber. For example, a microscope slide cover slip can be made from this glass (or borosilicate glass) using a method that yields a flat, uniform, smooth glass sheet.
[0040]
Alternatively, a further embodiment of the present invention uses a silica substrate. Silicon wafers used commercially in the semiconductor industry have a natural oxide layer by air oxidation. Such chips and surfaces offer the advantage of a high quality smooth silica surface that can be functionalized using methods similar to silicate glass.
[0041]
Other materials suitable for constructing the patch-clamp microchamber component include, but are not limited to, a polymer surface. Activation of the polymer surface can be performed using a UV / ozone or plasma process. Activation of the polymer surface also involves the creation of new chemical functional groups on the polymer surface caused by ionic and electron bombardment. The process is effective not only for increasing the wettability of the polymer surface but also for increasing the affinity of the charged bipolar lipid membrane on the polymer substrate.
[0042]
The present invention provides devices and methods for performing multi-channel patch clamp experiments. FIG. 2 shows a front perspective view of such an electrophysiological measurement device 10 according to one embodiment of the present invention. Apparatus 10 comprises a housing 12 having a pair of inlets 14 and 16 into which a multi-well plate (with an array of wells) can be inserted. One type of multi-well plate that can be used in accordance with one embodiment of the present invention is a multi-well plate having a plastic body and a solid glass bottom. One example of such a plate is model number 7706-2375, commercially available from Whatman Polyfiltronics. One of the plates may contain cells, while the other holds the solution to be transferred to the plate with cells. Located above the entrances 14 and 16 is a set of control buttons 18 for controlling operation of the device 10. For example, the control buttons 18 can be used to dispense cells into the wells of a multiwell plate, apply a pressure difference, create a voltage gradient, display various measured electrophysiological parameters, and the like. After evaluation, the multiwell plate may be removed from housing 12 and discarded.
[0043]
FIG. 3 is a cross-sectional view of the device 10 according to one embodiment of the present invention. The inlet 14 communicates with a generally open inner chamber 20 for holding a multi-well plate 22 having a plurality of wells 24 as shown in FIG. 4 (a similar inner chamber communicates with the inlet 16). ). When the plate 22 is placed in the inner chamber 20, it is held in a chamber 26 having a common electrode 28. In use, chamber 26 is filled with an electrolyte solution and current is provided through openings in each of wells 24 by applying a voltage to common electrode 28 as described herein. The common electrode 28 is connected to a control unit 29 having suitable electronic circuits for providing current to the common electrode 28.
[0044]
Dispense compound into each well using a dispenser. For example, a multiwell dispensing device 30 having a plurality of dispensing tips 32 is disposed above the inner substance 20. Each dispensing tip 32 is connected to a line 34 leading to a reservoir in the control unit 29. The solution in which the cells are suspended can be supplied to each dispensing tip 32, which then supplies the cells (hundreds to thousands of cells) in the solution to the wells 24 of the plate 22.
[0045]
Electrodes and electronics are provided to measure the electrophysiological properties of each cell or cell membrane tested in each well. For example, each dispensing tip 32 further includes a well electrode 36 that provides a path for return current from the common electrode 28. In other embodiments, the electrode is separate from the dispensing tip and may be, for example, integrated into the side of the well, or a thin film electrode on the side or end of the well. Good. Each well electrode 36 is connected to an electronic circuit in the control unit 29, and a voltage gradient is formed across the cell membrane of the cells deposited in each opening in the well 24. In addition, the control unit 29 includes electronic circuits suitable for measuring and recording changes in voltage and current for each cell membrane.
[0046]
The device may also have the ability to drain wells or chambers and maintain a positive or low vacuum to facilitate formation of a giga seal. A pressure difference is applied between each well 22 and the chamber 26 in order to capture cells inside from the opening of each well 24. While dispensing the cells into the wells, the chamber 26 can be pressure adjusted to a slightly positive or slightly negative pressure to assist in the formation of a gigaseal as described below. This can be done by applying a positive pressure through each of the dispensing tips 32 or by evacuating the chamber 26. The control unit 29 can control this vacuum.
[0047]
Control unit 29 further includes suitable electronic circuitry for recording and storing electrophysiological properties. The control unit 29 may include suitable input and output ports that allow this data to be electronically transferred to another computer or other storage device for future use.
[0048]
Still further, the control unit 29 may be used to lower the dispensing tip 32 into the well 24 after the plate 22 has been inserted into the inlet 14. After lowering the dispensing tip 32, the cells in the solution can be dispensed to each of the wells 24 using the control unit 29 as described above. When the operation is completed, the plate 22 can be automatically discharged from the inlet 14 using the control unit 29. Then, the plate can be taken out and discarded.
[0049]
FIG. 5 illustrates another embodiment of an electrophysiological measurement device according to one embodiment of the present invention. Apparatus 38 includes a housing 40 having an interior for holding a multi-well plate 42 having a plurality of wells 44. For convenience of illustration, only three wells are shown. However, it will be appreciated that the device 38 can be configured with a variety of well configurations. In one example, the well is about 800 μm deep and about 2 mm in diameter. Further, plate 42 need not be horizontal and may be oriented in other orientations. Below the plate 42, a chamber 46 for holding the electrolyte solution is arranged. In the chamber 46, a common electrode 48 formed of a metal plate is reciprocatably disposed. Electrodes 48 are connected to appropriate electronics so that a voltage gradient can be formed across the cell membrane, as described herein.
[0050]
Above the plate 42, a multiwell dispensing device 50 having a plurality of dispensing tips 52 is arranged. Dispensing device 50 is configured such that dispensing tip 52 is inserted into well 44 after plate 42 is inserted into device 38. The dispensing tip 52 may include a seal 54 to seal between the dispensing tip 52 and the well 44 when a pressure differential is applied to the well 44 as described herein. Each dispensing tip 52 further includes a well electrode 56. In this way, a voltage gradient can be formed between the common electrode 48 and the well electrode 56 when measuring the electrophysiological properties of the cell or cell membrane. The electrodes 56 are further connected to appropriate electronics as described in FIG. 5 so that voltage and current measurements can be collected and recorded.
[0051]
The end of each well 44 has a tapered through opening 58 that provides a current path between the common electrode 48 and the well electrode 56. With such a configuration, the cells 60 can be dispensed into the wells 44 using the dispensing device 50. The cells 60 are dispensed as an electrically conductive solution. Chamber 46 may also be filled with an electrically conductive solution such that a voltage gradient is formed across the cell membrane of the cells in each well 44.
[0052]
6-9 illustrate how to use the device 38 according to an embodiment of the present invention. FIG. 6 shows a more detailed view of one embodiment illustrating the openings in one of the wells of the screening device shown in FIG. Cells 60 in solution are dispensed into each well 44 using dispenser 38. The common electrode 48 includes a plurality of openings 62 corresponding to each of the through openings 58. First, the common electrode 48 can be shifted such that the opening 62 is offset from the through opening 58. At this time, the solution in the well 44 will not flow into the chamber 46.
[0053]
As shown in FIG. 7, the electrode 48 is translated so that the opening 62 and the through opening 58 are aligned. Thus, the solution in the well 44 flows into the chamber 46. Further, as shown, a pressure differential may be formed such that one of the cells 60 is drawn to the end of the through opening 58. Such a pressure differential can be created by supplying a positive pressure from the dispensing tip 52 and / or by evacuating the chamber 46. The magnitude of the pressure can vary depending on the type of seal formed between the cell 60 and the side of the through opening 58. For example, the sides of the through opening 58 may optionally include an adhesive-like material so that a high resistance seal is formed between the cell 60 and the side wall of the through opening 58. Such an adhesive is indicated by reference numeral 64 in the figure. Further, a pressure difference can be formed so that a gigaseal is formed between the cell 60 and the side wall of the through opening 58. Optionally, by forming a voltage difference between the electrodes to generate a potential difference, it can be confirmed whether an appropriate seal has been formed. If a suitable seal is not formed, exclude wells without a giga seal from consideration.
[0054]
As shown in FIG. 8, the electrode 48 can be translated to make a hole in the bottom of the cell 60 that extends below the through opening 58. Thus, in one embodiment, when the common electrode 48 returns to its original position and a voltage gradient is applied, as shown in FIG. 9, the interior of the cell 60 may be at the same potential as the common electrode 48.
[0055]
In any embodiments of the present invention, a "penetrated patch" is formed by cutting a portion of the cell wall surrounded by a high resistance seal or giga seal. This can be achieved, for example, by using a cutter located adjacent to the plate. The cutter allows one or more openings to be cut or formed in cells protruding below the ends of the wells. The common electrode may be configured to function as the cutter. The interior of the cell may be placed at the same potential as one of the common electrodes. Alternatively, the bottom of the cells can be pierced using pressure or electric pulses, or by using an orifice forming solution containing Nystatin or other antibiotics. Further, as an alternative to using the electrode 48 as a cutter, the device 38 may use a large pressure pulse to destroy the bottom of the cell 60, or use a Nystatin solution to form a hole in the bottom of the cell 60. it can.
[0056]
At the location shown in FIG. 9, the measurement of the electrophysiological properties can be performed by forming a voltage gradient and measuring the current passing through the ion channels of the cell membrane. As described above, by using the device 38, a plurality of cells or cell membranes can be evaluated in parallel with high throughput. After performing the measurement, the plate 42 can be taken out and discarded.
[0057]
FIG. 10 shows an embodiment of a multi-well plate 66 that can be used in any of the measurement devices of the present invention. Plate 66 is comprised of a plate body 68 having an “top” end 70 and a “bottom” end 72. A plurality of wells are formed in the plate body, and each well is open at an upper end 70. Further, as shown in FIG. 11, each well 74 has a “bottom” end 76. The plate body may be made of plastic while the bottom end 76 is made of glass. For example, a glass sheet may be adhered to the bottom of a polystyrene 96-well plate. Because of this, the plate 66 can be manufactured relatively inexpensively and can be discarded after use.
[0058]
11A-11F show an embodiment of a multi-well plate 66 that can be used in any of the measurement devices of the present invention in more detail. FIG. 11A shows a more detailed view of wells 74 of the multi-well plate of FIG. FIG. 11B shows a more detailed view of well 74 of FIG. FIG. 11C is a cross-sectional view of one of the wells 74 of the multi-well plate of FIG. FIG. 11D is a more detailed view, shown in detail D, of the through opening in the well of FIG. 11C. FIG. 11E is a more detailed view of the through opening of FIG. 11D, shown in detail E.
[0059]
According to embodiments of the present invention, several different types of holes having different geometries are used. In one embodiment (FIG. 11E), there is a conical opening 78 having a diameter of about 30 μm, the diameter of which is reduced to a through hole 80 of about 2-5 μm in diameter. Each bottom end 76 has an opening 78 for receiving a cell. The taper angle of the opening 78 and the size of the through hole 80 may be varied to optimize the giga seal depending on the cell type and the average cell size, for example, the taper angle is about 1 ° to 90 °. °. In one embodiment of the invention, the cells have a mean diameter of about 8 μm to 80 μm. In another embodiment, the cells have a mean diameter of between about 8 μm and 12 μm, alternatively between 10 μm and 12 μm.
[0060]
In one embodiment of the present invention, one technique for forming through-opening 78 is to use laser drilling or related techniques. The laser drilling method is a method in which a material is repeatedly irradiated with a pulse of focused laser energy and vaporized layer by layer until a through hole is formed. In this way, what is known as a "popped" or "percussion drilled" hole is formed. Depending on the material and the thickness of the material, the popped holes can be as small as 1 μm in diameter. If a larger hole (larger than 100 μm in diameter) is required, the laser can be moved along the desired diameter, once the material has been penetrated, along the working piece. The end result is a fast and efficient way to create high quality holes. Preferably, these holes are created using an ultraviolet laser, but infrared lasers can also be used. In addition, the laser drilling method can be automated to make the drilling of holes more precise and accurate.
[0061]
In FIG. 11F, the openings 85 in the plate 90 are substantially less conical in shape and only offset a few degrees from a line drawn perpendicular to the surface. Opening 85 further has a "large" end 86 and a "small" end 87, each having a different diameter 97 and 99, respectively. It is characterized by the point. In one embodiment of the present invention, the opening 85 has a diameter of about 7-9 μm on the large side 86 and about 1-3 μm on the small side 87. In this embodiment, the thickness 91 of the glass plate in FIG. 11F is about 100 μm.
[0062]
In one embodiment of the invention, as shown in FIG. 1, the larger end of the conical opening engages the cell and the smaller end is open to the chamber. In another embodiment of the present invention, the smaller end of the opening adheres to the cell because the large end and the small end are reversed.
[0063]
FIG. 12 shows another embodiment of the present invention, in which the holes are formed in two stages, a counterbore and a through hole. The counterbore (or blind hole) 100, which is a relatively large opening (having a diameter 102 of about 80-100 μm), is drilled halfway through a glass disc 105 100-120 μm thick using a mask. In a glass having a thickness of 100 μm, the depth 101 is about 80 to 100 μm. Alternatively, the counterbore 100 is drilled without using a mask. Subsequently, a second slightly conical "through hole" 115 is drilled into the approximately 15-20 μm glass remaining at the bottom of the counterbore hole to have a diameter of approximately 2 μm. The hole drilled by the laser drill in this embodiment is slightly tapered and has an angle of 2-5 °. This slight taper is due to the laser drilling method. The cells 120 are considerably smaller than the counterbore but larger than the opening of the through-hole, and are located on the through-hole when a gigaseal is formed.
[0064]
As described above, in one embodiment, the counterbore 100 and the through-hole 115 are drilled using a laser. The laser for counterbore 100 has a wavelength of 193 nm and the laser for through hole 115 has a wavelength of 248 nm. Alternatively, the laser has a wavelength of about 150-300 nm for both counterbore 100 and through-hole 115. The wavelength used to drill the holes is determined by the type of glass (or other material) used. Some glasses do not adequately absorb 248 nm, in which case a 193 nm laser can be used. In other embodiments, both the counterbore 100 and the through-hole 115 can be drilled using the same wavelength laser (eg, a 248 nm laser). When the same laser is used, the diameter of the laser beam can be changed using a different mask.
[0065]
FIG. 13A shows a perspective view of another embodiment of a multi-well plate (eg, a composite 96-well plate) according to one embodiment of the present invention. The glass plate or sheet 125 has a plurality of openings. The opening can be created using any of the methods described herein. The layer of plastic 130 (eg, silicone elastomer) also has a larger array of holes, which may be mounted on a glass plate or adhered. The resulting glass / plastic composite constitutes a multi-well plate according to the practice of the present invention.
[0066]
FIG. 13B is an exploded view of a multi-well plate including a glass plate and a plastic sheet, also showing a test vacuum fixture, according to one embodiment of the present invention. FIG. 13B also shows a lower chamber that further includes a vacuum fixture 135.
[0067]
FIG. 13C shows an enlarged perspective view of one well 121 of a multi-well plate according to one embodiment of the present invention. Well 121 comprises a plastic layer 130 adhered to a glass plate or sheet 125 having openings 140. Opening 140 may comprise a counterbore and a through hole. Therefore, the side surface of the well 121 is made of the plastic 130, and the bottom surface of the well 121 is made of the glass plate or sheet 125.
[0068]
FIG. 13D shows another enlarged perspective view of one well 121 according to one embodiment of the present invention. This “hidden line” view of well 121 more clearly shows a through hole having a top opening 145 and a bottom opening 150. Also in this case, the side surface of the well 121 is made of plastic 130, and the bottom surface of the well 121 is made of a glass plate or sheet 125.
[0069]
FIG. 14 illustrates one embodiment of a patch clamp microchamber according to one embodiment of the present invention. The device has a well 200 for receiving a solution 205 containing cells 210. In one embodiment, the well further comprises an electrode 215 made of silver coated with silver chloride. Although the electrodes are depicted in FIG. 14 as resting on plate 220, the electrodes may be connected to well sidewalls 225, as described herein, or other implementations. In an embodiment, it may be part of a liquid dispensing system as shown in FIG. The patch clamp microchamber according to FIG. 14 further comprises a chamber 230 for holding an electrically conductive buffer solution together with the second electrode 235. In one embodiment of the invention, the bottom of the chamber has a window 240 for inspecting the opening 245. Since the openings 245 receive the individual cells 210, measurements are taken on the individual cells 210 as described herein. Such measurements allow the measurement of the electrophysiological properties of cells or cell membranes, and are commercially available patch clamp recording electronics designed for pipette-based ion channel recording (eg manufactured by Axon Instruments or HEKA Elektronik) Recorded system).
[0070]
Referring further to FIG. 14, the patch clamp microchamber may further comprise a tube 250 extending from the chamber for controlling the pressure of the buffer solution inside the microchamber. In one embodiment, a vacuum source 255 is connected to the microchamber via a tube 250 to evacuate the chamber interior (eg, -1 to -5 kPa or -1 to -2 kPa). The cells are aspirated into openings 245 by vacuum (or negative pressure) to assist in forming a gigaseal.
[0071]
In other embodiments, positive pressure is applied from the same tube. Such a pressure differential blows the cleaning buffer through the opening, keeping the debris away from the opening until the cells approach the opening, thereby depositing cells into the opening and the surface surrounding the cell membrane and opening 222. And the formation of a giga seal between them is facilitated. Alternatively, positive pressure can be applied to the well through the opening when adding cells. FIG. 14 also illustrates a relatively short distance between cell electrodes 215 and 235, and an amplifier 260 that amplifies the input signal to provide a strong signal output 265.
[0072]
The patch clamp microchamber shown in FIG. 14 has wells for receiving one or more cells. In one embodiment, the volume of the wells of the patch-clamp microchamber device is minimal, necessary for test compound handling and cell viability. For example, the volume of a well is about 300 μL, and the bottom of the well has an opening several microns in diameter.
[0073]
The patch clamp microchamber can further comprise a liquid dispensing system having a dispenser configured to place cells in solution into the wells. In this case, cells can be supplied to the wells using, for example, an automatic robot. A first electrode is provided that can be located in the well. The well electrode may be associated with one or more dispensers such that placement and removal of the electrode is controlled by an automated robot. Each dispenser may include a seal member to form a seal with the well, so that a positive pressure can be applied to each well. Alternatively, a liquid dispensing system can dispense liquid into an array of addressable wells. At this time, each of the wells is independently addressable by an automatic liquid dispensing system. The wells can be addressed so that the dispensing system can identify individual wells and place specific cells (or cell types) in specific wells.
[0074]
In another embodiment, the patch clamp microchamber comprises a plate having a plurality of wells for receiving cells. The patch-clamp microchamber having a plurality of wells may further include a common chamber located adjacent to each opening used to hold an electrically conductive buffer solution. In one embodiment, a common electrode is disposed within the chamber and comprises a plurality of well electrodes located within the well and capable of forming a voltage gradient across the cell membrane of the cell within the opening. . In this way, the electrophysiological properties of multiple cells or cell membranes can be measured simultaneously. In other embodiments, individual wells each have a separate chamber.
[0075]
According to another embodiment of the present invention, there is provided a multi-channel liquid dispensing system having a plurality of dispensers that may be configured to place cells in solution into each well. Thus, cells can be supplied to each well using, for example, an automatic robot. In one embodiment of the invention, the well electrode may be connected to a dispenser such that the placement and removal of the electrode is controlled by an automated robot. Each dispenser may include a seal member to form a seal with the well, so that a positive pressure can be applied to each well.
[0076]
FIG. 15 illustrates one embodiment of a patch clamp microchamber with a glass disk, according to one embodiment of the present invention. The device comprises a well 300 for receiving a solution 310 containing cells 320. The well further comprises a glass disk 330 with an opening 340 separating the well from the chamber 350. The electrodes may be configured in multiple ways, for example, as shown in FIG. The glass disc 330 fits into the microchamber and has one or more openings 340. In one embodiment of the present invention, the glass disc 330 can be removed from the microchamber and used for another microchamber or array thereof. In one embodiment, a vacuum source 360 is connected to the microchamber through a tube 370, and the chamber can be evacuated. Such a vacuum facilitates the deposition of cells in the opening 340, forming a high resistance seal.
[0077]
The surface surrounding the opening of the laser drilled glass plate or sheet may be modified to facilitate the formation of a high resistance seal between the cell membrane and the surface of the opening in the well. Such modifications include, but are not limited to, heating the glass plate (eg, baking in an oven), bonding an adhesive-like substance to the surface of the plate surrounding the opening 222, or Covalently attaching the lipid to the surface of the plate surrounding the plate. Alternatively, the surface of the plate can be modified as described herein before drilling the openings in the wells.
[0078]
In one embodiment, the glass, glass disk, sheet or plate is heat treated prior to the experiment, for example, by heating the glass (eg, by baking in an oven) to or near the softening temperature of the glass. The softening temperature of a glass is the temperature at which the glass loses sufficient viscosity at which it stops behaving like a brittle solid and begins to flow like a liquid. For example, since borosilicate glass has a softening temperature of 736 ° C., the heat treatment can be performed at 700 ° C. for 3 to 10 minutes. The heating temperature and time can further vary depending on the hole geometry, dimensions (eg, counterbore depth, through-hole cone angle, etc.), glass type, and glass thickness. The heat treatment has the effect of modifying the surface surrounding the opening because the glass is heated from an external heat source and because the glass is thinnest near the opening. Heating the glass improves the quality of the giga seal. In one example, the actually used patch clamp microchamber with a counterbore and through-hole drilled by a laser drill as shown in FIG. 12 measured about 725 MΩ before heating. After heating, the resistance was a high value of 10 GΩ. After heat treatment or firing, the glass can be immediately incorporated into a microchamber for patch clamp experiments.
[0079]
The softening temperature of glass can be measured, for example, using the following method. The method can be found at "http://enterprise.astm.org/PAGES/C338.htm", which is incorporated by reference in its entirety;
1. This test method is based on a nominally round glass fiber with a diameter of 0.65 mm and a length of 235 mm with a specified tolerance, and the upper 100 mm of the length in a specified heating furnace at a rate of 5 + 1 ° C / min. It involves measuring the softening point of the glass by measuring the temperature at which it elongates at its own weight at a rate of 1 mm / min when heated.
[0080]
2. This standard does not intend to solve all of the security problems, if any, when using them. It is the responsibility of the user of this standard to take appropriate safety and health measures and to determine the application of pre-use regulatory restrictions.
[0081]
In one embodiment of the invention, the surface of the plate surrounding the opening is modified by applying an adhesive-like substance to the side wall or surface surrounding the opening. This can be done, for example, by immersing the bottom of the multi-well plate in a reservoir containing an adhesive-like substance and subsequently shaking the plate or applying a small pressure to one side of the plate to remove excess adhesive It can be done by doing. Similarly, other known techniques for applying the adhesive-like substance may be used. When dispensed into the wells, the cells form a tightly sealed interface (GigaSeal) with the wall of each well, allowing electrophysiological properties to be measured. Adhesive materials include silicone-based adhesives, vaseline / paraffin-based compositions, and the like. Such an adhesive material is preferably a chemically inert soft grease-like material. This allows cells to adhere to the surface of the through-opening to form a gigaseal. In one embodiment, a seal is formed that has a leakage resistance between about 600 megaohms and about 1.1 gigaohms.
[0082]
In another embodiment of the present invention, a surface coating of an adhesive-like substance or lipid can be placed on the side wall or surface surrounding the opening. Thus, multiple cells can be screened simultaneously by placing cells in individual wells where a high resistance seal is formed between each cell and the opening of each well. The formation of multiple seals by allowing the formation of multiple seals facilitates high-throughput screening when simultaneously evaluating multiple cells using electrophysiology techniques.
[0083]
FIG. 16 shows covalent attachment of lipids to a plate surface, such as glass, in one embodiment of the present invention. The cell membrane contains a lipid bilayer. Both layers of the lipid bilayer are composed of phospholipid molecules, each having a polar "head" and a non-polar "tail". In water, the phospholipid molecules are oriented such that their polar heads face the surrounding aqueous environment and the polar ends of the second layer are exposed to the aqueous environment of the cytosol. The non-polar ends of each layer are in contact and make up the "inside" of the cell membrane.
[0084]
The attachment of the phospholipid layer to the surface can be achieved by covalent or non-covalent bonds. For example, glass surfaces can be functionalized; for example, reactivity can be imparted by linking amino-silane moieties to silica and glass surfaces using methods known to those skilled in the art.
[0085]
Lipids having various functional groups suitable for covalently attaching to a modified surface are commercially available, for example, from Avanti Polar Lipids, Inc. In one aspect of the invention, such functionalized lipids can be used to attach to the modified surface of the invention. Methods for covalently linking a functionalized molecule to a molecule immobilized on a surface, for example, are known to those skilled in the art.
[0086]
In one embodiment of the invention, suitable complementary functional groups on suitable lipids include nucleophiles and carbon electrophiles. The terms "nucleophilic group" and "electrophilic group" have their ordinary meaning in synthetic and / or physical organic chemistry. Carbon electrophiles include one or more alkyl, alkenyl, alkynyl or aromatic (sp.) Substituted by any atom or group having a Pauling electronegativity greater than carbon itself.^Three, Sp^Two, Or sp-hybridized) carbon atoms. Examples of preferred carbon electrophiles include, but are not limited to, carbonyl (especially aldehydes and ketones), oximes, hydrazones, epoxides, aziridines, alkyl halides, alkenyl halides, and aryl halides, acyl, sulfonates (aryl, Alkyl and the like). Other examples of carbon electrophiles include unsaturated carbons that are electronically conjugated to electron withdrawing groups, such as β-carbon in α, β-unsaturated ketones or carbon in fluorine-substituted aryl groups. Is an atom. Generally, carbon electrophiles are susceptible to attack by complementary nucleophiles, including carbon nucleophiles. In that case, the aggressive nucleophile imparts an electron pair to the carbon electrophile such that a new covalent bond is formed between the nucleophile and the electrophile.
[0087]
In one embodiment of the present invention, suitable carbon electrophiles include carbonyl, epoxide, aziridine, cyclic sulfates and sulfamidates, and alkyl halides, vinyl halides and aryl halides. In one embodiment of the present invention, suitable nucleophilic groups include primary and secondary amines, thiols, thiolic acids, and thioethers, alcohols, alkoxides. These nucleophilic groups, when used in combination with a preferred carbon electrophile, typically form a heteroatom bond (CXC) between the homing peptides and the scaffold (where X Is a heteroatom such as oxygen or nitrogen).
[0088]
In another embodiment of the present invention, the phospholipid may comprise a photolabile functional group suitable for binding to a glass plate or other substrate when activated by light.
[0089]
Amine or phospholipids having an activated carbonyl function (eg, N-hydroxysuccinyl (NHS) ester) can be linked to a surface having a complementary function. For example, a lipid having an amine will react with a surface having NHS ester groups while forming an amide bond. Similarly, lipids with NHS esters will react with lipids with amines, again forming simultaneously amide bonds. The choice of complementary functional group permutation depends on the actual experimental conditions, such as cell type, presence of other components. One advantageous form of the invention is a modular approach for connecting partners. One skilled in the art can envision other complementary carbon electrophiles and nucleophiles capable of forming covalent bonds, which are within the scope of the invention. For example, a phospholipid having a thiol (-SH) functional group can be linked to a surface having a thiol (-SH) by forming a disulfide bond.
[0090]
Referring further to FIG. 16, glass surface 600 has hydroxyl groups 610 that can be functionalized with aminosilane 620 using known methods. Other reactive groups can be immobilized on the glass surface by other methods known in the art. Other functionalized glass surfaces suitable for linking to functionalized lipids include aldehydes, epoxides, maleimides, nickel chelates, streptavidin, biotin, thiols, and the like.
[0091]
The aminosilane shown in FIG. 16 has five --CH_TwoWhile having a hydrocarbon linker with a-group, those skilled in the art will appreciate that the actual linker length may vary. In FIG. 16, the end of the aminosilane is an amino group 630 suitable for covalent attachment to a lipid molecule 640 having a complementary electrophilic functional group 650. The resulting linkage creates a new covalent bond 660. This surface chemical reaction results in a high density of lipid molecules on the glass surface.
[0092]
FIG. 17 illustrates the covalent attachment of lipid molecules 700 to a surface 710 surrounding an opening 720 in a patch clamp device according to one embodiment of the present invention. In this embodiment, one end of each lipid molecule is attached to the surface, and the other end is free to interact with the cell membrane. In one embodiment of the present invention, such a covalently bonded layer may be a film. Thus, a gigaseal is formed between the surface and the cell.
[0093]
Phospholipids can be selectively bound to the surface surrounding the pore. In one embodiment of the present invention, holes can be drilled in a plate or substrate by laser drilling prior to covalently attaching lipids. The lipids can then be selectively linked using photoreactive functional groups. Other areas of the plate or disk can be photomasked.
[0094]
Chemical treatment with liquids can also be used to increase the affinity of cell membranes for polymer surfaces. One example is exposure to a caustic soda solution. The alkaline solution hydrolyzes the ester groups on the polymer surface to increase wettability and also increases the surface affinity for the charged bipolar lipid membrane.
[0095]
Various embodiments of the patch clamp devices disclosed herein include a pressure control system. 18A and B illustrate the formation of a giga seal according to one embodiment of the present invention using, for example, a pressure control system. The glass plate 800 has an opening 810. The geometry of the opening may be of any of the types disclosed herein. A positive pressure 820 is applied to the bottom chamber while filling the top chamber with a solution 840 containing cells. Without being bound by any particular theory, such conditions are such that an expanded area or "bubble" of clean intracellular fluid emanating outwardly 830 from the pores toward the wells containing the cells. Has the effect of forming If there are microfluidic properties, the expansion bubbles of the cleaning fluid are formed without disturbing. Experiments have shown that the cleaning liquid can flow through the pores without affecting the formation of the gigaseal, which then occurs when cells adhere to the opening.
[0096]
Normal healthy cells have a higher protein content than dead or diseased cells and are therefore denser than cell debris and dead cells. Because healthy cells are denser, they sink faster than dead or diseased cells. Agglomerates of cells, which can be as dense as healthy cells, have a higher ratio of hydrodynamic drag to weight than healthy or `` good '' single cells, which is also higher. It tends to sink slowly. As the bubble of the cleaning liquid 830 expands, cell debris and cell clumps are carried away further than "good" cells. Thus, "bubbles" can carry cell debris and cell clumps farther away from the pores than "good" cells, while the cell mass sinks to the bottom.
[0097]
Once the proper configuration has been established (FIG. 18B), suction or negative pressure 850 is applied to deflate the "bubble" of clean liquid. As the bubble collapses, "good" cells 860 are drawn into the pores before cell debris and cell clumps. In this way, a gigaseal is formed and the "good" cells remain inside the bubble 860. On the other hand, dead or diseased cells and cell debris remain outside the bubble 850.
[0098]
The following step-by-step protocol is an example of a protocol that can be used to reliably form a giga seal using any of the patch clamp devices disclosed herein. First, install the patch clamp chamber and properly incorporate the electrodes. Second, dispense approximately 100 μL of cells at 5 million cells / mL into the upper well. Here, about 10 μL of Ringer's solution (solution inside the pipette) is added to the well in advance. Third, pressure is applied to the bottom chamber. In one embodiment, the pressure is applied using a 10 mL syringe. Compressing the syringe from 10 mL to 5 mL results in a pressure of about 14 psi. Next, release the plunger. The plunger typically slowly returns to 9.8 mL, which is about 0.3 psi. Fourth, wait one minute. One minute is a good example of the length of time to wait, as previous experiments have shown that cells generally settle in one minute. Fifth, suction is performed at a pressure of -5 kPa.
[0099]
The above procedures and parameters may be optimized for particular cells and other experimental variables. High success rates of gigaseal formation can be achieved with this procedure; an overall success rate of 80% can be achieved. In other embodiments, the devices and techniques of the present invention facilitate the formation of a gigaseal between cells and openings in the wells of a multi-well plate.
[0100]
The invention further provides a method for automating the screening of ion channel assays, allowing many compounds and many cells to be processed in parallel at a time. The present invention can utilize natural cell lines, for example, CHO, Jurkat, HEK, and hERG. The present invention allows the same compound to be screened against multiple targets in the same experiment with increased throughput.
[0101]
Multichannel allows the same drug molecule to be screened against multiple target ion channels in the same experiment. When used as a drug discovery tool, the present invention can be used to determine whether a drug is a good modulator of an ion channel. The present invention allows for maximum flexibility and control over the components of each well. In the device, the components of each well, such as cell type, drug candidate, buffer, ionic concentration, etc., may be different depending on the experiment being performed.
[0102]
The present invention allows for the use of ion channels as "biosensors". In addition to acting directly on gating ion channels, drugs can act on other molecular targets in cells and on ion channel activity. The effects observed by measuring electrophysiological properties using the devices and methods disclosed herein may be correlated with the mechanism of drug activity, and may include, for example, the efficacy of a particular drug. Can be quantified in terms of drug concentration. Such accurate measurements are useful when comparing or distinguishing drug candidates from each other.
[0103]
In one embodiment of the present invention, drug-induced regulation of protein kinases and phosphatases in cells that alter the kinetic behavior of specific ion channels is achieved by using highly accurate electrical and electronic methods using the devices and methods disclosed herein. It can be detected and recorded by a physiological assay. In addition, several cell lines can be used to assess the effect of the same drug on specific kinases, phosphatases, the phosphorylation of ion channels, and the effect of the drug on the channel itself.
[0104]
In other embodiments, the device can be used to measure accurate pH changes. The proton-gated ion channel is a proton (H⁺Or alternatively a hydronium ion (H_Three ⁺O)) only selective. Such a proton-selective ion channel is a natural pH meter. This is because when two types of solutions having different ion concentrations are separated by a selective membrane such as an ion channel, the concentration can be measured as a potential difference. Furthermore, if such differences exist, the ions will move to eliminate the imbalance. This flow can be observed as an ionic current.
[0105]
One commercially available pH meter shows that the semipermeable glass membrane used has other ions, such as sodium ions (Na⁺), The accuracy is low. Because proton ion channels are selective for protons, pH meters based on ion channels improve on existing situations in the art. Thus, the present invention can serve as a substitute for a microscopic physiological function measuring instrument.
[0106]
In another embodiment of the present invention, the devices and methods disclosed herein can be used to construct "proton biosensors" for drug discovery. When certain drugs are introduced into cells, intracellular pH may change. In still other embodiments, the device itself may function as a pH meter, for example, if the drug cannot cross cell membranes. In this case, the measured pH change corresponds to the pH of the solution in the well.
[0107]
The output of electrophysiological information from a single experiment of the present invention may involve multiple parameters recorded essentially simultaneously. The technique of the present invention also allows the cytoplasm of the cells to be dialyzed. Thereby, it becomes possible to adjust the composition of the intracellular fluid by introducing or removing specific ions from the intracellular fluid. This method allows one to evaluate one of the channels while dialysis of the cytoplasm of the cell in an experiment, excluding other types of channels. This allows one channel to be optimized while excluding the others. In addition, electrophysiological methods have a high degree of selectivity and are capable of recording the activity of a single channel molecule. The technology of the present invention also has a high temporal resolution (in the sub-millisecond range) and is useful for targets of some ion channels, such as the Na channel, which deactivates quickly.
[0108]
Since the ion current passing through the ion channel is on the order of a few picoamps (pA), it is difficult to measure accurately. In one embodiment of the present invention, electrical noise pickup is minimized by making the distance between the cell electrode and the amplifier relatively short (eg, a few millimeters). In one embodiment of the present invention, the distance is necessary and sufficient to connect the electrode inside the well to the chip. Thus, a chip-based amplifier can clearly convert a pA signal to a mV signal. A common multi-channel data acquisition board can acquire this signal without difficulty.
[0109]
In yet another aspect, an electronic circuit is provided for measuring a voltage value and / or a current value for each well. Also provided is a liquid dispensing system and a controller for controlling operation of the electronic circuit. Further, a voltage source is connected to the common electrode to form a voltage gradient.
[0110]
In another embodiment, the invention provides a method for measuring current through an ion channel of a plurality of cells. The method utilizes a plate with a plurality of wells each having an end. At least some of the wells have openings at the ends, the chamber is located below the plate, and is filled with an electrolyte solution. A common electrode is also located in the chamber. In such a configuration, the cells are dispensed into the solution in the well. The pressure differential between the well and the chamber collects the cells at the opening, creating a high resistance seal between the cells and the end of the well. A potential difference is provided between the common electrode and the well electrode located in each well. Measurements of electrophysiological properties are obtained from cells or cell membranes located within the openings. Thus, a high-throughput screening system can be created by evaluating a plurality of cells in parallel. Alternatively, the cells are deposited in the chamber and then drawn into the opening such that only a portion of the cells are in the opening. Measurements can then be obtained as described above by penetrating a portion of the cells extending into the chamber.
[0111]
The present invention further provides a method of measuring current through an ion channel of a cell. The method utilizes at least one well having an end and a sidewall within the end that defines an opening through the end of the well. An adhesive-like substance is placed on the side wall of the opening and one or more cells are deposited in the opening. An adhesive is used to form a high resistance seal between the cells and the side walls that form the openings. A potential difference is then formed across the cell membrane and voltage and / or current measurements are collected and recorded. Thus, such a method creates a high resistance seal sufficient to make the electrophysiological measurement accurate. Further, such techniques allow the use of simple and inexpensive multi-well plates constructed of plastic or glass instead of silicon and nitride, or multi-purpose plates made of glass.
[0112]
One example of a procedure for performing a screening experiment is expressing a target ion channel in a cell line. Only one cell per well needs to be measured, but each well is configured to receive several of these cells. The plate is placed on a chamber containing the intracellular fluid. The common electrode located in the chamber may consist of a metal plate that can move so that the solution can flow down from each well. Further, it will be appreciated that one or more common electrodes can be used. For example, two or three common electrodes can be used. By applying a slight pressure or applying a vacuum to each well, cells can block the through-opening, thereby closing the opening. Such a procedure would take approximately 1-3 minutes for cells to be able to form a high resistance seal with the hole at the end of each well.
[0113]
The metal plate can then be moved back and a hole can be drilled in the lower part of the cell that has passed through each well by the applied pressure. Alternatively, cells can be punctured with a pressure pulse or a perforation solution. As another alternative, cells can be penetrated by electroporation. Once the hole has been drilled in the lower part of the cell, the system is ready to record electrophysiological properties with high throughput.
[0114]
If a suitable seal is formed, a voltage with a voltage difference of about -70 mV will be formed between the intracellular electrode (a common electrode made of a metal plate) and each needle located in the well. In this example, the voltage is negative because the reference is defined outside the cell.
[0115]
Before collecting measurements, each well can be tested to determine if a seal has been formed. If not, the well can be labeled as a well with a "bad" seal (without a giga seal) and excluded from further consideration. The plate can be tested multiple times during the experiment to again confirm the stability of the seal formation. Each well can be tested by applying a small hyperpolarizing pulse to the cell membrane. By excluding wells without a gigaseal from further discussion, ligand is effectively saved because the ligand is applied only to "good" wells.
[0116]
Subsequently, voltage and current measurements of a single cell can be collected and recorded using conventional patch clamp electronics. The recorded data is stored and evaluated to determine the efficacy of the test compound. Furthermore, cells can be evaluated at high throughput.
[0117]
19A and 19B illustrate two methods of using SQUIDs to measure ionic current, according to one embodiment of the present invention. SQUID is a highly sensitive magnetic field sensor. SQUID is an abbreviation for "Superconducting Quantum Interference Device". With SQUID, small changes in the magnetic field can be measured with high accuracy and very high sensitivity. Improved versions of SQUID are also suitable for measuring magnetic field gradients, voltages, currents, and susceptibility.
[0118]
The output voltage of SQUID is sinusoidal as a function of the applied magnetic flux. Therefore, its behavior is non-linear and periodic. One period coincides with the magnetic field quantum Φ, where Φ₀= 2 x 10^-15Wb. When an input coil is used as a current-to-magnetic-flux converter, very high-sensitivity current amplification can be performed. Another way to increase the sensitivity of SQUID is to use multiple SQUIDs in series. When n SQUIDs are operated in phase, the output voltage increases in proportion to n, but the noise of the SQUID increases more slowly in proportion to the square root of n.
[0119]
One method of using a SQUID according to one embodiment of the present invention is to pass the current generated by the ion channel through a wire, thereby generating a magnetic field that the SQUID detects as shown in FIG. 19B. Such a detector has the advantage that the measurement of the current and the voltage stimulation can be decoupled. Using a pneumatic electrical switch, the detector can be automatically switched to a different cell without additional electrical noise. Multiplexing can be realized, for example, by sequentially recording a plurality of wells using a recording device. Multiplexing is a term used to describe the use of inputs that can switch to multiple sources and sample measurements sequentially. Each recording device can read from one well. Further, multiplexing can be performed in parallel by a plurality of recording devices.
[0120]
In addition, cables can be mechanically plugged into suitable sockets using aligned pneumatic cylinders for noise-free connections. Switch the compressed air far away from the detector. The location of the switch can be very close to the cells, to minimize the length of the cable, and also to minimize the capacitance and inductance of the cable.
[0121]
In one embodiment of the present invention, another method of using a SQUID is to measure the magnetic field when the channel is active. The SQUID sensor can sense a weak magnetic field formed by the current of the ion channel when placed near a cell. In one embodiment, as shown in FIG. 19A, an hourglass-shaped capillary holding a cell or SQUID sensor surrounds an opening on a flat substrate or plate.
[0122]
In FIG. 19A, the SQUID device 400 is configured to detect a change in magnetic flux by a loop 410 surrounding the opening 420, ie, the path of the ion current. In this embodiment, the SQUID device functions independently of the circuit having the two electrodes 430 and 440 and the measuring device 450.
[0123]
FIG. 19B shows a SQUID device 500 configured to detect a change in magnetic flux by a loop 510 surrounding a wire 520. In this embodiment of the invention, the SQUID device detects a signal that can be physically disconnected from the ion channel and / or cell chamber.
[0124]
The steps depicted for the methods herein may be performed in a different order than depicted and / or mentioned. The steps herein are merely exemplary of the order in which the steps can be performed. The steps herein may be performed in any desired order, and even in such cases, the objects of the claimed invention will still be achieved. Furthermore, steps that are not desired to be used can be eliminated from the steps in the method of the invention, and in such cases the object of the claimed invention is still achieved.
[0125]
All patents and publications mentioned herein are hereby incorporated by reference to the same extent as if each individual patent or publication was specifically and individually incorporated by reference.
[0126]
Those skilled in the art will appreciate that the present invention is well suited to achieving the stated purposes as well as the results and technical advantages, as well as those inherent to them. The particular systems and methods described herein as presently representative of the preferred embodiments are exemplary, and are not intended to limit the scope of the invention. Those skilled in the art will recognize those modifications and other uses that fall within the spirit of the invention as defined by the appended claims.
[0127]
It will be apparent to those skilled in the art that modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention described herein by way of description as suitable may be practiced in the absence of any factors and limitations not specifically disclosed herein. The terms and expressions used are used as terms of description and not for purposes of limitation, and are not intended to be limiting, but rather include any components or parts thereof shown or described in using such terms and expressions. While it is not intended to exclude equivalents, it is recognized that various modifications may be made within the scope of the claimed invention. Thus, while the present invention is specifically disclosed by preferred embodiments and optimal configurations, those skilled in the art can take steps to modify and modify the concepts disclosed herein; It is to be understood, however, that such modifications and changes are considered to be within the scope of the invention as defined by the appended claims.
[0128]
In addition, where the features or aspects of the present invention are described in the form of Markush groups or other optional groups, those skilled in the art will recognize that the present invention is not intended to limit the individual members or members of the Markush groups or other groups. Will be described by the sub-group of For example, if options A, B and C are present, all of the following possibilities are included: A only, B only, C only, A and B, A and C, B and C, and A and B and C.
[0129]
Thus, additional embodiments exist within the scope of the invention and the appended claims.
[Brief description of the drawings]
[0130]
FIG. 1 illustrates an apparatus for measuring electrophysiological properties according to one embodiment of the present invention.
FIG. 2 is a front perspective view of a high-throughput screening apparatus according to an embodiment of the present invention.
FIG. 3 is a schematic sectional view of the screening apparatus of FIG. 2;
FIG. 4 is a front perspective view of a multi-well plate used in combination with the screening device of FIG. 3;
FIG. 5 is a view showing another embodiment of the electrophysiological measuring device according to one embodiment of the present invention.
FIG. 6 is a more detailed view of an opening in one of the wells of the screening device of FIG.
FIG. 7 illustrates the screening device of FIG. 6 after cells have been drawn into the opening, according to one embodiment of the present invention.
FIG. 8 illustrates a common electrode translated to ablate a portion of a cell closing a through-opening, according to one embodiment of the present invention.
FIG. 9 illustrates a common electrode when it returns to its original position where measurements of electrophysical properties can be made, according to one embodiment of the present invention.
FIG. 10 is a top perspective view of one embodiment of a multi-well plate that can be used in a high-throughput screening device, according to one embodiment of the present invention.
FIG. 11 is a plan view of the multi-well plate of FIG.
11A is a more detailed view of one well of the multi-well plate shown in FIG. 11, with detail A extracted. FIG.
FIG. 11B is a more detailed view of the well shown in FIG. 11A, with detail B extracted.
FIG. 11C is a cross-sectional view of one of the wells of the multi-well plate shown in FIG.
FIG. 11D is a more detailed view of detail D of the through-opening in the well shown in FIG. 11C.
FIG. 11E is a more detailed view of the through-opening shown in FIG. 11D, with detail E extracted.
FIG. 11F illustrates another embodiment of a through-opening in the plate that is substantially less conical than the configuration of FIG. 11E.
FIG. 12 is a diagram showing a two-stage opening composed of a counterbore and a through hole.
FIG. 13A is a perspective view of another embodiment of a multi-well plate according to one embodiment of the present invention.
FIG. 13B is an exploded view of a multi-well plate including a glass plate and a plastic sheet, also showing a test vacuum fixture, according to one embodiment of the present invention.
FIG. 13C is an enlarged perspective view of one well of a multi-well plate according to one embodiment of the present invention.
FIG. 13D is another enlarged perspective view of one well according to one embodiment of the present invention.
FIG. 14 illustrates a patch clamp microchamber according to one embodiment of the present invention.
FIG. 15 illustrates a patch clamp microchamber with a glass disk according to one embodiment of the present invention.
FIG. 16 illustrates covalent attachment of lipids to a plate surface according to one embodiment of the present invention.
FIG. 17 illustrates binding of lipid molecules near an opening in a patch clamp device according to one embodiment of the present invention.
FIG. 18A illustrates the formation of a giga seal in accordance with one embodiment of the present invention.
FIG. 18B illustrates the formation of a giga seal according to another embodiment of the present invention.
FIG. 19A is a diagram illustrating a patch clamp device having a SQUID detector according to one embodiment of the present invention.
FIG. 19B is a diagram showing a patch clamp device having a SQUID detector according to another embodiment of the present invention.

Claims (88)

An apparatus for measuring an electrophysiological property of a cell membrane of an individual cell,
A plate having at least one opening surrounded by a surface that is modified to facilitate formation of a giga seal;
A chamber adjacent the plate in fluid communication with at least one of the openings and adapted to hold a solution;
It comprises a first electrode located in the chamber, and a second electrode located adjacent to the plate,
The above device for use in measuring the electrophysiological properties of cell membranes of individual cells. The apparatus of claim 1, further comprising an amplifier in electrical contact with both electrodes. The device of claim 1, wherein cells or cell membranes adhere to the surface of the opening to form a gigaseal. The apparatus of claim 1, wherein the plate has wells, and some of the wells are replaceable or replaceable. The apparatus of claim 4, wherein the replaceable portion is a disc having an opening. 5. The device of claim 4, wherein the sides of the well are made of plastic and the bottom of the well is made of glass. The device of claim 1, wherein modifying the surface comprises chemically modifying a surface surrounding the at least one opening. 9. The device of claim 7, wherein chemically modifying comprises covalently attaching the substance to the plate. The device of claim 1, wherein the substance is covalently attached to a surface of the well surrounding the opening. The device of claim 1, wherein modifying the surface comprises modifying a surface surrounding the opening by a heat treatment. The apparatus of claim 10, wherein the plate comprises glass, and wherein the heat treatment comprises heating the surface to or near the softening temperature of the glass. The device of claim 1, wherein at least one opening is tapered. The apparatus of claim 1, wherein at least one opening has a counterbore and a through hole. The device of claim 1, wherein the cells are in solution and the plate has wells, further comprising a multi-channel liquid dispensing system having a plurality of dispensers configured to place the solution in wells. The above device. The apparatus of claim 1, further comprising a vacuum source coupled to the chamber for evacuating the interior of the chamber. The apparatus according to claim 1, further comprising an electronic circuit for measuring a voltage value and / or a current value for each well. 17. The device of claim 16, further comprising a SQUID detector. The apparatus of claim 1, wherein the plate comprises a multi-well plate having an array of wells each having an opening. 20. The device of claim 18, further comprising an automated liquid dispensing system independently addressable to each well. The device of claim 1, wherein the electrophysiological properties of the cell membrane are recorded by measuring the current flowing through the first and second electrodes. The apparatus of claim 1, wherein at least one electrode comprises silver having a silver chloride coating. The device of claim 1, wherein the solution is an electrically conductive solution. The apparatus of claim 1, wherein the aperture is created using a laser. An apparatus for measuring an electrophysiological property of a cell membrane of an individual cell,
A plate having at least one well with an opening made using a laser and modified by heating to receive individual cells;
A chamber adjacent the plate, in fluid communication with the opening, and adapted to hold an electrically conductive solution;
A first electrode located in the chamber,
A second electrode located in the well, and comprising an amplifier in electrical contact with the first electrode and the second electrode,
The above device for recording the electrophysiological properties of the cell membrane of individual cells by measuring the current flowing through the first and second electrodes. 25. The device of claim 24, wherein the opening has a counterbore and a through hole. 26. The device of claim 25, wherein the counterbore is drilled to a depth of about 80-110 [mu] m. 26. The device of claim 25, wherein the through holes have a diameter of about 2-5 [mu] m. 26. The apparatus of claim 24, further comprising a vacuum source coupled to the chamber for evacuating the interior of the chamber. The device of claim 24, further comprising a SQUID detector. 25. The apparatus of claim 24, wherein the plate comprises a multi-well plate having an array of wells each having an opening. 31. The apparatus of claim 30, further comprising an automated liquid dispensing system that can independently address each well. 25. The apparatus of claim 24, wherein the plate has wells, the sides of the wells are made of plastic, and the bottoms of the wells are made of glass. A removable disk having an opening that functions as part of a well for use in measuring electrophysiological properties of a cell membrane. 34. The disk of claim 33, comprising a glass. 34. The disc of claim 33 having a plurality of openings. 34. The disc of claim 33, wherein the surface surrounding the opening is chemically modified. The disk according to claim 33, wherein the surface surrounding the opening is heat-treated. 38. The disc of claim 37, wherein the disc comprises glass, and wherein the heat treatment includes heating the surface to or near the softening temperature of the glass. 38. The disc of claim 37, wherein the heating comprises laser heating. 34. The disk of claim 33, wherein the opening has a counterbore and a through hole. 41. The disk of claim 40, wherein the size of the counterbore is about 130 [mu] m and the size of the through hole is about 2 [mu] m. Providing at least one well with an opening having a modified surface for receiving cells having a cell membrane;
Depositing cells in the openings, forming a gigaseal between the cells and the wells with the modified surface, and recording voltage and / or current measurements to assess cell membrane ion channels. , A method of measuring the current passing through an ion channel of a cell membrane. 43. The method of claim 42, wherein the sides of the well are made of plastic and the bottom of the well is made of glass. 43. The method of claim 42, further comprising applying a vacuum using a vacuum source to assist in forming a giga seal. 43. The method of claim 42, further comprising using an automated liquid dispensing system to add cells, buffers and test compounds. 43. The method of claim 42, wherein modifying the surface comprises modifying a surface surrounding the opening by a heat treatment. 47. The method of claim 46, wherein at least one well is on a plate, wherein the plate comprises glass, and wherein the heat treatment comprises heating the surface to or near the softening temperature of the glass. 43. The method of claim 42, wherein modifying the surface comprises chemically modifying a surface surrounding the at least one opening. 43. The method of claim 42, wherein the openings are created using a laser. 43. The method of claim 42, wherein at least one opening has a counterbore and a through hole. 51. The method of claim 50, wherein the counterbore is created using a laser having a wavelength of about 150-300 nm. 51. The method of claim 50, wherein the through holes are created using a laser having a wavelength of about 150-300 nm. Providing at least one well having an opening;
Placing a solution containing a plurality of cells into a well;
Applying a positive pressure to the opening and applying a negative pressure to the opening to draw one of the plurality of cells into the opening to form a gigaseal between the cell and the opening. A method of forming a giga seal. 54. The method of claim 53, further comprising recording voltage and / or current measurements to assess an ion channel of a cell membrane of one of the plurality of cells. 54. The method of claim 53, wherein the opening is surrounded by a surface, the surface being modified to assist in forming a gigaseal. 54. The method of claim 53, wherein said one of said plurality of cells comprises good cells. 54. The method of claim 53, wherein the sides of at least one well are made of plastic and the bottom of at least one well is made of glass. 56. The method of claim 55, wherein the surface has been modified by a heat treatment. 59. The method of claim 58, wherein at least one well is on a plate, wherein the plate comprises glass, and wherein the heat treatment comprises heating the surface to or near the softening temperature of the glass. 54. The method of claim 53, wherein the opening has a counterbore and a through hole. 54. The method of claim 53, wherein the opening is created using a laser. At least one well having an end and a sidewall within the end defining an opening;
Glue placed on the side of the opening, such that a high resistance seal is formed between the cell and the side wall,
A first electrode, which may be located in the well, and the exterior of the well, which allows a voltage gradient to be formed across the membrane of the cell, which is located in the opening, so that electrophysiological measurements of the cell membrane can be recorded. An apparatus for facilitating electrophysiological measurement of a biological material, comprising a second electrode that may be located at 63. The device of claim 62, wherein the adhesive is configured to form a high resistance seal having a leakage resistance of about 600 megaohms to about 1.1 gigaohms. 63. The device of claim 62, wherein the adhesive comprises a silicone-based adhesive. A plate having the wells, together with a plurality of other wells each defining an opening at an end,
A chamber adjacent to the plate in fluid communication with each of the holes and adapted to hold an electrically conductive solution, and a set of first electrodes that can be located in each of the other wells;
63. The device of claim 62, wherein the second electrode comprises a common electrode located within the chamber. A plate having a plurality of wells each having an end, wherein a hole is formed at an end of at least a part of the wells, the hole receives each cell, and a high-resistance seal is formed on the cell. And a plate configured to be formed between the and the end,
A chamber adjacent to the plate in fluid communication with each of the holes and adapted to hold an electrically conductive solution;
A common electrode;
A plurality of well electrodes configured to be located inside the well for forming a voltage gradient across a membrane of the cell located within the pore so that electrophysiological measurements of the cell can be collected. A device that facilitates the electrophysiological measurement of biological material. 67. The device of claim 66, wherein each hole is tapered. 68. The device of claim 67, wherein the narrowest dimension of each hole is in a range from about 1 [mu] m to about 5 [mu] m. 67. The device of claim 66, wherein each aperture comprises an adhesive adapted to form a seal between the wall of the aperture and the cell. 67. The apparatus of claim 66, further comprising a multi-channel liquid dispensing system having a plurality of dispensers configured to place cells in solution into the wells. 71. The device of claim 70, wherein the well electrode is connected to a dispenser. 67. The apparatus of claim 66, further comprising a vacuum source coupled to the chamber for evacuating the interior of the chamber. 71. The apparatus of claim 70, wherein each dispenser further comprises a seal member for forming a seal with the well such that a positive pressure can be supplied to each well. 71. The apparatus according to claim 70, further comprising electronic circuitry for measuring a voltage value and / or a current value for each well. The apparatus of claim 74, further comprising a controller for controlling operation of a liquid dispensing system and the electronic circuit. 67. The apparatus of claim 66, further comprising a voltage source coupled to the common electrode. 67. The device of claim 66, further comprising means for creating a small hole in the cell. 78. The apparatus of claim 77, wherein the means for creating a hole is selected from the group consisting of a reciprocable common electrode, a pressurized solution, and a hole forming solution. A support means comprising cell retaining means, wherein said cell retaining means is adapted to receive individual cells such that a high resistance seal is formed between said cell retaining means and said cells. Support means having
Means for storing an electrically conductive liquid arranged below the support means,
An ion channel for a biological material comprising means for forming a voltage gradient across the cell membrane in each pore, and means for collecting and recording measurements of cell voltage and / or current. A device for facilitating evaluation. 80. The device of claim 79, further comprising an adhesive means for forming a high resistance seal between the cell and the cell holding means. Providing at least one well having an end and a sidewall within the end defining an opening;
Placing an adhesive on the side walls of the opening,
Depositing cells in the openings, where a high-resistance seal is formed between the cells and the sidewalls with an adhesive;
A method for measuring current through an ion channel of a cell, comprising forming a potential difference across a cell membrane and recording a voltage and / or current measurement. Providing a plurality of wells having sidewalls defining openings, depositing an adhesive on each sidewall, depositing cells in each well, and forming a potential difference across each cell membrane to provide a voltage and / or voltage; 82. The method of claim 81, further comprising recording a current measurement. A plate having a plurality of wells each having an end, wherein at least some of the wells have holes formed at the ends of the wells, and a chamber adjacent to the plate and filled with an electrolyte solution And providing a common electrode,
Dispensing cells in the solution into wells,
Creating a pressure differential between the well and the chamber to collect the cells in the pores and form a high resistance seal between the cells and the end of the well;
Placing a well electrode in the well,
Creating a potential difference between the common electrode and a well electrode disposed in the well, and collecting electrophysiological measurements of cells located in the pores;
A method for measuring a current passing through an ion channel of a plurality of cells, comprising: 84. The method of claim 83, further comprising testing whether a suitable seal has been formed between the cell and the end of the well. 84. The method of claim 83, wherein the high resistance seal is at least about 1 gigaohm. 84. The method of claim 83, further comprising placing an adhesive in the hole to form a seal between the cell and the end of the well. 87. The method of claim 86, wherein the adhesive forms a high resistance seal between about 600 megaohms and about 1.1 gigaohms. 84. The method of claim 83, wherein the electrophysiological measurements of the cells are collected at the same time that the cells are dispensed into the wells.

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