Pipette Guidance in Multiwell Plate Patch Clamp

Abstract

A patch clamp system and associated processes are provided that include a multi-well patch clamp plate and manifold and can provide improved fluid control in patch clamp experiments.

Description

[Technical field]

[0001] A patch clamp system and associated processes are provided. The present invention provides improved fluid control in patch clamp experiments performed on multi-well patch clamp plates. [Background technology]

[0002] Patch clamp systems and methods are used to monitor the electrophysiological properties of ion channels in ion channel-containing structures, typically lipid membrane-containing structures such as cells, by establishing an electrophysiological measurement configuration in which the cell membrane forms a high resistance seal around a measurement electrode, allowing the flow of electrical current through the cell membrane to be determined and monitored. Such systems can form part of an apparatus for carrying out patch clamp techniques utilized, for example, to study ion transport channels and biological membranes.

[0003] The general idea of ​​electrically isolating a patch of membrane and studying ion channels within that patch under voltage clamp conditions has been reviewed in the literature (Neher, Sakmann, and Steinback (1978) "The Extracellular Patch-clamp, A Method For Resolving Currents Through Individual Open Channels In Biological Membranes", Pfluger Arch. 375; 219-278). Recent developments in patch clamp techniques contemplate the introduction of planar substrates (e.g., silicon chips) instead of traditional glass micropipettes (see, e.g., WO 01 / 25769 and Mayer, 2000). Additional background art includes U.S. Pat. No. 8,268,260, which is incorporated herein by reference.

[0004] Sophion Biosciences' commercial instrument, QPatch, performs fully automated patch clamp measurements. However, there is a need for a higher degree of fluid control than is possible with fully automated systems. In particular, while manual pipetting of fluids into the patch clamp plate offers the user greater flexibility, patch clamp systems typically require careful fluid addition to avoid excessive flow effects and ensure even distribution of the agent under test. Further complications can arise from variability in individual user pipetting technique.

[0005] European Publication No. 2681550 describes substrates for patch clamp analysis. US Publication No. 2011 / 0251102 describes multi-well plates and patch clamp apparatus. Summary of the Invention [Problem to be solved by the invention]

[0006] Thus, one object of the present invention is to provide a patch clamp system and related processes based on a multi-well patch clamp plate that allows improved liquid control. In particular, the present technique allows for guiding the pipetting of liquids into such a patch clamp plate. Advantageously, the patch clamp system has a simple structure and is easy to use. In particular, the moving parts in the system should be limited in terms of complexity. [Means for solving the problem]

[0007] A patch clamp system is provided, the patch clamp system comprising a patch clamp plate and a manifold, the patch clamp plate comprising a plurality of intracellular (IC) inlets arranged in a first array, each IC inlet being fluidly connected to an IC chamber, a plurality of extracellular (EC) inlets arranged in a second array, each EC inlet being fluidly connected to an EC chamber, and a patch clamp substrate arranged between each EC chamber and each IC chamber, each patch clamp substrate comprising at least one patch clamp hole extending through the patch clamp substrate and providing a fluid connection between the EC chamber and the IC chamber, each IC chamber comprising an IC electrode, and each EC chamber comprising an EC electrode.

[0008] The manifold comprises a plurality of first pipetting channels arranged in a third array, and optionally a plurality of second pipetting channels arranged in a fourth array.

[0009] The manifold is movable between a first position and a second position relative to the patch clamp plate, wherein in the first position, each of the first or second pipetting channels of the manifold is configured to be aligned with one IC inlet of the patch clamp plate, and in the second position, each of the first or second pipetting channels of the manifold is configured to be aligned with one EC inlet of the patch clamp plate.

[0010] The system allows guided pipetting of liquids at required locations within a plate by moving a manifold between a first and a second position.

[0011] A process for priming and a process for performing patch clamp measurements are also provided.The present invention further provides a patch clamp apparatus comprising a patch clamp system. [Brief description of the drawings]

[0012] Further details of the technology are provided in the attached dependent claims, figures and examples.

[0013] [Figure 1] FIG. 1 is a schematic cross-sectional view of a patch clamp plate according to the present invention. [Diagram 2] FIG. 1 shows a general perspective view of a patch clamp plate as viewed from above (i.e., the main surface). [Diagram 3] FIG. 1 shows a general perspective view of the patch clamp plate and manifold from above. [Figure 4] A cross-sectional view of the patch clamp plate and manifold in position ready for IC pipette injection is shown. [Diagram 5] A cross-sectional view of the patch clamp plate and manifold in position ready for EC pipette injection and pressure control is shown. [Figure 6A] FIG. 1 shows a cross-sectional view of the patch clamp plate, manifold and pipette guide in position ready for IC pipette injection. [Figure 6B] A cross-sectional view of the patch clamp plate, manifold and pipette guides in position ready for EC pipette injection and pressure control is shown. [Figure 7A] With the pipette tip in place, a cross-sectional view of the patch clamp plate, manifold and pipette guide in position, ready for IC pipette injection, as in FIG. 6A. [Figure 7B] With the pipette tip in place, a cross-sectional view of the patch clamp plate, manifold and pipette guide in position ready for EC pipette injection and pressure control as in FIG. 6B. [Figure 8] FIG. 1 shows a schematic diagram of an experimental setup equipped with a patch clamp system of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] The patch clamp technique is used to study ionic currents within individual isolated live cells, tissue slices, or patches of cell membrane. This technique has been used to study neurons, cardiomyocytes, muscle fibers, and pancreatic beta cells, among others.

[0015] Various patch clamp techniques are used: in voltage clamp, the voltage across the cell membrane is controlled and the resulting current is recorded, and in current clamp, the current through the membrane is controlled by the user and the resulting voltage changes are recorded.

[0016] A patch clamp system is provided that includes a patch clamp plate and a manifold. In general, the patch clamp plate captures and holds cells while performing patch clamp experiments, and the manifold controls air pressure and guides various pipetting procedures.

[0017] (Patch clamp plate) Patch clamp plates are used in automated patch clamp (APC) measurements for high-throughput recording of ion channel currents in live cells. Patch clamp plates are assembled from multiple components with a range of properties that are advantageous for APC measurements.

[0018] Patch clamp plates are typically "single-use" (disposable), i.e., they are manufactured in a straightforward manner from disposable materials (mainly molded plastics) so that they can be discarded after each use, reducing or eliminating contamination between experiments.

[0019] The patch clamp plate comprises a plurality of intracellular (IC) inlets arranged in a first array, each IC inlet being fluidly connected to an IC chamber. The patch clamp plate also comprises a plurality of extracellular (EC) inlets arranged in a second array, each EC inlet being fluidly connected to an EC chamber.

[0020] The layout of the first and second arrays are suitably identical, so that adjacent IC inlets in the first array are separated by the same distance and in the same direction as adjacent EC inlets in the second array.

[0021] Suitably, the IC inlets in the patch clamp plate are arranged along a first axis (AA) in a first linear array, and the EC inlets in the patch clamp plate are arranged along a second axis (BB) in a second linear array, preferably the first axis (AA) and the second axis (BB) are parallel. This method simplifies alignment of the pipetting channels in the manifold with the EC / IC inlets in the plate.

[0022] The patch clamp plate also includes a patch clamp chip (typically made of silicon, but optionally also made of plastic) disposed between each EC chamber and each IC chamber, each patch clamp chip including one or more patch holes extending through the patch clamp chip and providing a fluid connection between the EC chamber and the IC chamber. During a patch clamp experiment, biological cells to be examined are captured on the patch holes.

[0023] Each IC chamber of the patch clamp plate is equipped with an IC electrode and each EC chamber is equipped with an EC electrode.

[0024] The patch clamp plate may further comprise a cover, which shields the testing environment and provides an inlet for liquid access to the experimental site. A flexible gasket may be present to form a liquid and electrical seal between the EC and IC chambers. The IC and EC electrodes, which are suitably Ag / AgCl electrodes, are typically embedded in a ceramic substrate. Once assembled, the components form two microfluidic channels located above and below the patch site, which house the extracellular and intracellular solutions, respectively. The liquid inlets and outlets of the channels are located far away from the patch site to protect them from contamination.

[0025] The patch clamp plate suitably has a major surface, said major surface comprising a plurality of intracellular (IC) inlets arranged in a first array and a plurality of extracellular (EC) inlets arranged in a second array. If the IC and EC inlets are arranged on the same surface of the patch clamp plate, pipetting access is simplified as pipetting can be performed from the same surface. Typically, in use, the patch clamp plate is arranged with the major surface facing upwards and the manifold is arranged above this major surface.

[0026] The glass surface at the experimental site allows for the formation of gigaohm (GΩ) hermetic seals between the cell membrane and the patch hole using physiological solutions, allowing users to avoid "seal-enhancing" ions such as fluoride, which are often required in APC experiments and have been reported to unnecessarily modulate ion channel function.

[0027] The microfluidic channels connecting the extracellular fluid inlets to the patch sites are suitably covered with a glass layer, which minimizes compound adsorption, thereby ensuring reliable compound concentrations in concentration-response experiments.

[0028] Microfluidic channels on the outside of the cell allow rapid liquid exchange along with the addition of multiple compounds to the same cell during an experiment. In the design shown, solution exchange can be accomplished using less than 10 μL of solution, significantly reducing compound consumption. Laminar flow within the microfluidic channels prevents compound dilution upon addition and allows for complete washout of compounds subsequently, eliminating concentration corrections that can cause overshoot responses.

[0029] A patch clamp plate typically contains eight measurement sites, with each "measurement site" comprising an IC chamber, an EC chamber, a patch clamp tip, and associated EC / IC inlets and outlets.

[0030] The embedded electrodes (IC or EC electrodes) are suitably disposable Ag / AgCl electrodes. This ensures a high degree of measurement stability without the need for chlorination as known from manual patch clamp systems. The embedded electrodes in the patch clamp plate allow for a voltage offset (dV off / dt~0.005mV / min) and site resistance (dR site This allows for stable measurements over time with minimal drift (dt ~ 0.003 MΩ / min). The electrodes are positioned so that they can contact one or more electrical contacts in the patch clamp system, allowing patch clamp measurements to be performed.

[0031] The patch clamp plate typically further comprises a plurality of IC pressure relief outlets arranged in a sixth array, each of which is fluidly connected to an IC chamber, and a plurality of EC pressure relief outlets arranged in a seventh array, each of which is fluidly connected to an EC chamber, the pressure relief outlets being open to the pressure system in use.

[0032] Each patch clamp plate may further include a computer readable code, such as, for example, a bar code or QR code, to ensure plate and experiment traceability.

[0033] A commercially available patch clamp plate suitable for use in this technique is available from Sophion Bioscience A / S under the trade name "QPIate."

[0034] The patch clamp plate is preferably held in a holder, most clearly illustrated in FIG. 2. The holder aids in hygienic and safe manipulation of the patch clamp plate and in holding it in the correct position within the patch clamp system.

[0035] (Manifold) In a patch clamp system, a manifold is positioned adjacent to the patch clamp plate. The manifold can be used to selectively cover or expose the EC and IC inlets of the patch clamp plate and can guide various pipetting procedures. Suitably, the manifold can implement pressure protocols via the EC and IC inlets in the patch clamp plate. Furthermore, the relative alignment of the manifold to the patch clamp plate can be automated.

[0036] The manifold is suitably formed from a single piece of metal (e.g. aluminium, etc.) with suitable pipetting channels. The pipetting channels are suitably through channels in the manifold, tapering from a wide opening distal to the patch clamp plate to a narrower opening adjacent to the patch clamp plate, following the general tapering of a standard laboratory pipette tip. Each pipetting channel is designed to engage (i.e. contact) a standard laboratory pipette tip, for example to contact the outer wall of the pipette tip. Safe and accurate positioning of the pipette tip can thus be achieved (suitably at least in depth and also in the plane of the patch clamp plate).

[0037] Typically, the manifold comprises a plurality of first pipetting channels arranged in a third array, the third array of first pipetting channels suitably being identical to the layout of at least one of the first and third arrays of IC / EC inlets in the patch clamp plate, such that when the manifold and patch clamp plate are overlapped, the first pipetting channels in the first array can be aligned with the EC inlets in the second array and / or the IC inlets in the first array.

[0038] It may be sufficient for the manifold to consist of only one array of first pipetting channels (i.e., the third array), and the manifold movements can then be designed to achieve the correct EC / IC liquid and cell priming through the required EC / IC inlets.

[0039] Conveniently, the manifold may comprise a plurality of second pipetting channels arranged in a fourth array. The fourth array of second pipetting channels is suitably identical to the layout of at least one of the first and third arrays of IC / EC inlets in the patch clamp plate. The use of the third and fourth arrays of pipetting channels allows different liquids to be pipetted separately into the patch clamp plate, suitably to different depths within the patch clamp plate.

[0040] The manifold is moveable relative to the patch clamp plate. Suitably the manifold is located adjacent a major surface of the patch clamp plate and is moveable relative to the patch clamp plate at least in a plane parallel to said major surface.

[0041] In particular, the manifold is movable between a first position and a second position relative to the patch clamp plate. In the first position, each of the first pipetting channels or each of the second pipetting channels of the manifold is configured to be aligned with one IC inlet of the patch clamp plate. Thus, in the first position, the manifold guides the pipetting of IC liquid into the IC chamber via the IC inlet.

[0042] In the second position, each of the first pipetting channels or each of the second pipetting channels of the manifold is configured to align with one EC inlet of the patch clamp plate such that in the second position, the manifold guides the pipetting of EC liquid into the EC chamber via the EC inlet.

[0043] In a particular embodiment, the manifold comprises a plurality of first pipetting channels arranged in a third array and a plurality of second pipetting channels arranged in a fourth array, in which in the first position, each of the second pipetting channels of the manifold is configured to be aligned with one IC inlet of said patch clamp plate (100), and in the second position, each of the first pipetting channels of the manifold is configured to be aligned with one EC inlet of the patch clamp plate. The use of the third and fourth arrays of pipetting channels arranged as described above allows different liquids to be pipetted separately into the patch clamp plate, suitably to different depths within the patch clamp plate.

[0044] The patch clamp system may further comprise a pipette guide arranged such that the manifold is located between the pipette guide and the patch clamp plate (i.e., on the side of the manifold opposite the patch clamp plate). The pipette guide comprises a plurality of guide channels.

[0045] In particular, the pipette guide comprises a plurality of first guide channels, each of which is arranged to align with a first pipetting channel of the manifold, and a plurality of second guide channels, each of which is arranged to align with a second pipetting channel of the manifold. Suitably, the pipette guide is temporarily or permanently fixed to the manifold, for example by screwing, welding, adhesive, or other suitable means.

[0046] The pipette guide offers improved possibilities for adjusting the alignment and penetration depth of the pipette tip relative to the patch clamp plate, especially when temporarily fixed, e.g. by screwing, to the manifold.

[0047] In the patch clamp systems presented herein, the manifold, optionally together with pipette guides, may be configured to enable deeper and controlled penetration of a pipette tip into a patch clamp plate via multiple second pipette channels than the same pipette tip into a patch clamp plate via multiple first pipette channels.

[0048] In one arrangement, the manifold comprises opposing first and second surfaces, with a first pipetting channel and an optional second pipetting channel extending through the manifold from said first surface to said second surface. At least in said first and second positions, the first surface of the manifold is arranged to contact a major surface of the patch clamp plate. Suitably, in this arrangement, the manifold is rigidly clamped against the patch clamp plate in the first and second positions. A gasket may be arranged to provide an air-tight seal between the manifold and the patch clamp plate.

[0049] Preferably, when the manifold is in a position between said first and second positions, the first surface of the manifold does not contact the major surface of the patch clamp plate, i.e., it is elevated away from the major surface of the patch clamp plate, such that movement between the first and second positions is easily permitted and the risk of damage to the manifold and / or patch clamp plate is reduced.

[0050] The manifold may further comprise one or more IC pressure channels arranged in a fifth array. The fifth array should have the same layout as the first array of IC inlets in the patch clamp plate. The IC pressure channels are thus arranged such that in the second position, each of the IC pressure channels of the manifold is configured to align with each of the IC inlets in the first array. The IC pressure channels are used to (a) remove air trapped in the IC chamber and (b) provide a slight negative pressure in the IC chamber to fix the cell to the patch clamp hole. Thus, in the second position, each of the IC pressure channels of the manifold is configured to apply a gas pressure or a gas negative pressure to each of the IC inlets in the first array.

[0051] The manifold may further comprise one or more EC pressure channels. The EC pressure channels are arranged such that in the second position, the EC pressure channels of the manifold are configured to align with each EC pressure relief outlet of the patch clamp plate. The EC pressure channels are also used to (a) remove air trapped in the EC chamber and (b) apply a slight overpressure in the EC chamber compared to the IC chamber to immobilize the cell against the patch clamp hole. Thus, in the second position, the EC pressure channels of the manifold are configured to apply a gas pressure or a gas negative pressure to each EC pressure relief outlet.

[0052] If the manifold further comprises a plurality of pressure channels, the patch clamp system may further comprise at least one pressure controller in fluid communication with each of the IC pressure channels and / or each of the EC pressure channels in the manifold and configured to increase or decrease the pressure in each of the IC pressure channels and / or each of the EC pressure channels in the manifold, such that when the manifold is in the second position, the air pressure in the IC chamber and the EC chamber can be increased or decreased.

[0053] (Actuator) As mentioned above, the manifold and the patch clamp plate are displaceable relative to one another. The patch clamp system therefore suitably comprises an actuator, preferably attached to the manifold, which is capable of moving the manifold between said first and second positions. The actuator is configured to drive the manifold between said first and second positions relative to the patch clamp plate, preferably in a plane parallel to at least a major surface of the patch clamp plate. The actuator may also be arranged to drive the manifold in a second plane perpendicular to said major surface and preferably parallel to said first axis (AA) and second axis (BB) or including said first and second axes.

[0054] For simplicity, the actuator is preferably a linear actuator. The actuator is suitably arranged to clamp a first surface of the manifold against a major surface of the patch clamp plate in said first and second positions, thereby providing an air-tight seal.

[0055] The patch clamp plate is suitably mounted in a holder (H) for ease of manipulation, which may be a reusable component that can be inserted into and removed from the instrument.

[0056] (Priming process (pre-treatment)) The manifold allows for individual pressure control on the cell side of the patch hole. This allows precise control of priming and whole-cell formation in an adaptive manner, which increases the success rate of the experiment. The priming protocol ensures complete evacuation of air from the microfluidic channels before the start of the experiment.

[0057] Cells are applied through the extracellular channel and positioned at the patch hole by low negative pressure across the patch hole. If properly sealed, a short suction pulse places the cell in a whole-cell configuration and is held in place by low negative pressure during the subsequent patch clamp experiment. The extracellular solution can be exchanged throughout the experiment, allowing precise addition and washing of compounds. The channel design allows solution exchange using volumes as low as 3 μL, limiting compound consumption. The volume of the waste reservoir at the channel outlet allows a total of approximately 250 μL of solution to be added during the experiment.

[0058] Thus, a process for priming a patch clamp system is provided, the process comprising the steps of: -Preparing the patch clamp system as described herein. With the manifold in a first position, pipetting IC fluid into each intracellular (IC) chamber of the patch clamp plate via the IC inlet using a first pipetting channel in the manifold as a pipette guide. Moving the manifold to a second position. o Pressurize each IC chamber via one or more IC pressure channels in the manifold. o Pipetting the EC fluid into each extracellular (EC) chamber of the patch clamp plate via the EC inlet using the first pipetting channel in the manifold as a pipette guide. o Pressurizing each EC chamber via one or more EC pressure channels in the manifold.

[0059] Suitably, the manifold is clamped against the patch clamp plate in the second position.

[0060] After the priming process, cell capture may be performed, which includes pipetting a biological cell (C) into each extracellular (EC) chamber of the patch clamp plate through the EC inlet using a first pipetting channel in the manifold as a pipette guide while the manifold is in the second position, and then applying negative pressure through the patch hole to capture the biological cell at the patch hole.

[0061] (Patch clamp measurement) As mentioned above, patch clamp systems are used to perform patch clamp measurements on biological cells.

[0062] Thus, there is provided a process for determining and / or monitoring the electrophysiological properties of ion channels in biological cells in a patch clamp system as defined herein, said process comprising the steps of: - Providing a patch clamp system as defined herein. - Priming the patch clamp system according to the priming process defined herein. trapping one or more biological cells in said one or more patch holes according to a process defined herein. performing electrophysiological measurements on said biological cells.

[0063] There is also provided a patch clamp apparatus suitable for a laboratory setting, comprising a patch clamp system as defined herein, and further comprising a computer system having software, the computer system and associated software being configured to perform the following operations: o Controlling the pressure control device. o Controlling the actuators. o Controlling one or more patch clamp experimental parameters (e.g. cell stimulation). o Measuring electrical signals generated by a cell captured with a patch clamp system via the IC electrode and the EC electrode.

[0064] The device further comprises a user interface for communicating with a user of said device, said user interface being in electronic communication with said computer system, suitably in the form of a graphical user interface displayed on a computer display, preferably a touch screen display.

[0065] Suitably the apparatus comprises a housing within which said patch clamp system and said computer system are disposed.

[0066] Detailed Description of the Drawings 1 is a schematic cross-sectional view of a patch clamp plate 100 according to the present invention. An intracellular (IC) inlet 11 is shown fluidly connected to an IC chamber 12, and an extracellular (EC) inlet 21 is fluidly connected to an EC chamber 22.

[0067] The IC inlets and the EC inlets open onto the same (major) surface 101 of the plate 100. The EC chambers are located above (i.e. closer to the major surface 101) the IC chambers. Different liquids can be pipetted into the EC / IC chambers via the respective EC / IC inlets.

[0068] A patch clamp substrate 25 (here in chip form) is placed between the EC chamber 22 and the IC chamber 12. The patch clamp substrate 25 includes a patch clamp hole 26 onto which a cell (C) is trapped and held in place by a slight low pressure within the IC chamber 12. The patch clamp hole 26 extends through the patch clamp substrate 25 and provides a fluid connection between the EC chamber and the IC chambers 22,12.

[0069] The IC chamber 12 is equipped with an IC electrode 19, while the EC chamber 22 is equipped with an EC electrode 29. Patch clamp measurements on a cell C are essentially performed by measuring the current / voltage between the IC / EC electrodes.

[0070] FIG. 2 shows a general perspective view of the patch clamp plate as viewed from above (i.e., the first side). Each IC / EC inlet and IC / EC chamber, together with the associated IC / EC electrode and patch clamp substrate 25, constitutes a "site." FIG. 2 shows that in the patch clamp plate, multiple intracellular (IC) inlets 11 are arranged in a first array 10, and multiple extracellular (EC) inlets 21 are arranged in a second array 20. In FIG. 2, the IC inlets 11 are arranged along a first axis (AA), and the EC inlets 21 are arranged along a second axis (BB). FIG. 2 also shows a holder H into which the patch clamp plate can be inserted and removed.

[0071] FIG. 3 shows a general perspective view of the patch clamp plate and manifold from above, illustrating their relative arrangement. As shown, the manifold 50 covers a major surface 101 of the patch clamp plate 100. The manifold 50 has a plurality of first pipetting channels 51 arranged in a third array 52 and a plurality of second pipetting channels 53 arranged in a fourth array 54. The first pipetting channels 51 and the second pipetting channels 53 extend through the manifold 50 from a first surface 58 (not visible in FIG. 3) to a second surface 59, the first surface 58 of the manifold 50 being arranged in contact with the major surface 101 of the patch clamp plate 100. In FIG. 3, adjacent second pipetting channels overlap somewhat.

[0072] 4-7 show the functionality of the patch clamp system, and in particular the movement of manifold 50 relative to patch clamp plate 100. FIG.

[0073] 4 shows the manifold 50 in a first position relative to the patch clamp plate 100. In this position, the second pipetting channel 53 can be seen to be aligned with the IC inlet 11. IC liquid can now be pipetted into the IC chamber.

[0074] In the second position (shown in FIG. 5), the first pipetting channel 51 is aligned with the EC inlet 21. Pipetting of EC liquid and cells can be performed into the EC chamber. At the same time, in this second position, the pressure channel 55 of the manifold 50 is aligned with the IC inlet 11. The manifold can be clamped in this position, forming a seal via a gasket. Various pressure protocols can be performed via the pressure channels, and once a cell is trapped in the patch hole, the system is ready to perform a patch clamp experiment.

[0075] Figures 6A and 6B are similar to Figures 4 and 5 and further show a pipette guide 60 having a first guide channel 61 and a second guide channel 62. Adjustment of the pipette guide (or replacement of the pipette guide with a different pipette guide) allows pipetting at different depths and to accommodate different pipette sizes.

[0076] 7A and 7B show how manifold 50 and pipette guide 60 allow deeper penetration into patch clamp plate 100 via second pipette channel 53 than the same pipette tip can have via first pipette channel 51. This helps ensure proper pipette guidance and positioning, and optimal addition of liquid to the liquid interface of the EC and IC chamber fluids.

[0077] FIG. 8 shows a schematic diagram of a laboratory fixture 200 including a patch clamp system of the present invention, showing the housing 201 and where the patch clamp plate 100 is placed.

[0078] The present invention has been described with reference to several embodiments and figures. However, those skilled in the art can select and combine various embodiments within the scope of the present invention as defined by the appended claims. All documents referenced herein are incorporated herein by reference.

[0079] (Aspect) The following list of numbered aspects is provided: (Aspect 1) Patch clamp system The patch clamp system comprises a patch clamp plate (100) and a manifold (50). The patch clamp plate (100) comprises: a plurality of intracellular (IC) inlets (11) arranged in a first array (10), each IC inlet (11) being in fluid communication with an IC chamber (12); a plurality of extracellular (EC) inlets (21) arranged in a second array (20), each EC inlet (21) being fluidly connected to an EC chamber (22); a patch clamp substrate (25) disposed between each EC chamber (22) and each IC chamber (12), each patch clamp substrate (25) having at least one patch clamp hole (26) extending through the patch clamp substrate (25) and providing a fluid connection between the EC chamber and the IC chamber (22, 12); Each IC chamber (12) includes an IC electrode (19). Each EC chamber (22) includes an EC electrode (29). The manifold (50) is a plurality of first pipetting channels (51) arranged in a third array (52); Optionally, there is provided a plurality of second pipetting channels (53) arranged in a fourth array (54). The manifold (50) is movable between a first position and a second position relative to the patch clamp plate (100). In the first position, each of the first pipetting channels (51) or each of the second pipetting channels (53) of the manifold (50) is configured to be aligned with one IC inlet (11) of the patch clamp plate (100). In the second position, each of the first pipetting channels (51) or each of the second pipetting channels (53) of the manifold (50) is configured to be aligned with one EC inlet (21) of the patch clamp plate (100).

[0080] (Aspect 2) 2. A patch clamp system according to claim 1, wherein the manifold (50) comprises a plurality of first pipetting channels (51) arranged in a third array (52) and a plurality of second pipetting channels (53) arranged in a fourth array (54); In the first position, each of the second pipetting channels (53) of the manifold (50) is configured to be aligned with one IC inlet (11) of the patch clamp plate (100); In the second position, each of the first pipetting channels (51) of the manifold (50) is configured to be aligned with one EC inlet (21) of the patch clamp plate (100).

[0081] (Aspect 3) 11. The patch clamp system of claim 10, further comprising a pipette guide arranged such that a manifold is disposed between the pipette guide and the patch clamp plate, The pipette guide (60) is a plurality of first guide channels (61), each of which is arranged to be aligned with a first pipetting channel (51) of the manifold (50); Optionally, a plurality of second guide channels (62), each arranged to be aligned with a second pipetting channel (53) of the manifold (50).

[0082] (Aspect 4) A patch clamp system according to any one of aspects 2 to 3, wherein the manifold (50), optionally together with the pipette guide (60), is configured to enable deeper and controlled penetration of a pipette tip into the patch clamp plate (100) via the multiple second pipette channels (53) than the same pipette tip into the patch clamp plate (100) via the multiple first pipette channels (51).

[0083] (Aspect 5) 11. The patch clamp system of claim 10, wherein the patch clamp plate has a major surface, The main surface (101) includes a plurality of intracellular (IC) inlets (11) arranged in a first array (10) and a plurality of extracellular (EC) inlets (21) arranged in a second array (20).

[0084] (Aspect 6) A patch clamp system as described in aspect 5, wherein the manifold (50) is positioned adjacent to a major surface (101) of the patch clamp plate (100) and is movable relative to the patch clamp plate (100) at least in a plane parallel to the major surface (101).

[0085] (Aspect 7) 7. The patch clamp system of claim 6, wherein the manifold (50) comprises opposing first and second surfaces (58 and 59); a first pipetting channel (51) and, optionally, a second pipetting channel (53) extending through the manifold (50) from said first surface (58) to said second surface (59); In at least the first and second positions, a first surface (58) of the manifold (50) is positioned in contact with a major surface (101) of a patch clamp plate (100).

[0086] (Aspect 8) A patch clamp system according to any one of the preceding aspects, wherein the plurality of IC inlets (11) in the patch clamp plate (100) are arranged along a first axis (AA) in a linear first array (10); A plurality of EC inlets (21) in the patch clamp plate (100) are arranged along a second axis (BB) in a second linear array (20), preferably with the first axis (AA) and the second axis (BB) being parallel.

[0087] (Aspect 9) 13. The patch clamp system of claim 12, further comprising an actuator arranged to drive a manifold between the first and second positions relative to the patch clamp plate, Preferably, said actuation is at least in a plane parallel to said main surface (101), and more preferably, the actuator (90) is a linear actuator.

[0088] (Aspect 10) 10. The patch clamp system of embodiment 9, wherein the actuator is also arranged to drive the manifold in a second plane; The second plane is perpendicular to said main surface (101) and is preferably parallel to or includes the first axis (AA) and the second axis (BB).

[0089] (Aspect 11) A patch clamp system according to any one of aspects 9 to 10, wherein the actuator (90) is arranged to clamp a first surface (58) of the manifold (50) against a main surface (101) of the patch clamp plate (10) at the first position and the second position.

[0090] (Aspect 12) 20. The patch clamp system of claim 19, further comprising: a plurality of IC pressure relief outlets (80) arranged in a sixth array (81), each IC pressure relief outlet (80) in fluid communication with an IC chamber (12); A seventh array (83) includes a plurality of EC pressure relief outlets (82) arranged in the seventh array (83), each EC pressure relief outlet (82) fluidly connected to an EC chamber (22).

[0091] (Aspect 13) A patch clamp system as described in any one of the preceding aspects, wherein the manifold (50) further comprises one or more IC pressure channels (55) arranged in a fifth array (56), the IC pressure channels (55) being arranged such that, in the second position, each of the IC pressure channels (55) of the manifold (50) is configured to be aligned with each IC inlet (11) of the first array (10).

[0092] (Aspect 14) 20. The patch clamp system of claim 19, wherein the manifold further comprises one or more EC pressure channels; The EC pressure channels are arranged such that, in the second position, the EC pressure channels of the manifold (50) are configured to align with each EC pressure relief outlet (82).

[0093] (Aspect 15) The patch clamp system according to any one of the thirteenth to fourteenth aspects further comprises at least one pressure control device (70); The at least one pressure control device (70) is fluidly connected to each of the IC pressure channels (55) and / or each of the EC pressure channels in the manifold (50) and is configured to increase or decrease the pressure within each of the IC pressure channels (55) and / or each of the EC pressure channels of the manifold (50).

[0094] (Aspect 16) The patch clamp system of any one of the preceding aspects, further comprising a gasket disposed to provide an air-tight seal between the manifold (50) and the patch clamp plate (10).

[0095] (Aspect 17) The patch clamp system according to any one of the preceding aspects, wherein the patch clamp substrate (25) is a silicon substrate or a polymer substrate.

[0096] (Aspect 18) 11. A process for priming a patch clamp system according to any one of the preceding aspects, the process comprising: - providing a patch clamp system according to any one of the preceding aspects; With the manifold (50) in a first position, pipetting IC fluid into each intracellular (IC) chamber (12) of the patch clamp plate (100) through the IC inlet (11) using a second pipetting channel (53) in the manifold as a pipette guide; moving the manifold (50) to a second position; o pressurizing each IC chamber via one or more IC pressure channels in a manifold; o pipetting EC fluid into each extracellular (EC) chamber (22) of the patch clamp plate (100) via the EC inlet (21) using a first pipetting channel (51) in the manifold as a pipette guide; o pressurizing each EC chamber via one or more EC pressure channels in the manifold.

[0097] (Aspect 19) A process for trapping one or more biological cells in a patch clamp system according to any one of claims 1 to 17, comprising: The process comprises the priming process according to embodiment 18, pipetting biological cells (C) into each extracellular (EC) chamber (22) of the patch clamp plate (100) through the EC inlets (21) using a first pipetting channel (51) in the manifold as a pipette guide, with the manifold (50) in a second position; applying negative pressure through the patch hole (26) to capture the biological cells at the patch hole (26).

[0098] (Aspect 20) A process for determining and / or monitoring electrophysiological properties of ion channels in biological cells in a patch clamp system according to any one of the preceding aspects, the process comprising: Providing a patch clamp system according to any one of aspects 1 to 17; priming the patch clamp system according to the process of embodiment 18; capturing one or more biological cells in the one or more patch holes (26) according to the process of aspect 19; performing electrophysiological measurements on the biological cells.

[0099] (Aspect 21) A patch clamp device (200), comprising a patch clamp system according to any one of aspects 1 to 17, the device (200) comprising: a computer system having software, said computer system and associated software comprising: o controlling the pressure control device (70); o controlling an actuator (90); o controlling one or more patch clamp experimental parameters; o measuring an electrical signal generated by a cell captured in the patch clamp system via the IC electrode (19) and the EC electrode (29); The device (200) further comprises: a user interface for communicating with a user of the device, the user interface being in electronic communication with the computer system; Optionally, a housing (201) in which said patch clamp system and said computer system are disposed.

Claims

1. A patch clamp system comprising a patch clamp plate (100) and a manifold (50); The patch clamp plate (100) comprises: a plurality of intracellular (IC) inlets (11) arranged in a first array (10), each IC inlet (11) being fluidly connected to an IC chamber (12); a plurality of extracellular (EC) inlets (21) arranged in a second array (20), each EC inlet (21) being fluidly connected to an EC chamber (22); a patch clamp substrate (25) disposed between each EC chamber (22) and each IC chamber (12), each patch clamp substrate (25) having at least one patch clamp hole (26) extending through the patch clamp substrate (25) and providing a fluid connection between the EC chamber and the IC chamber (22, 12); Each IC chamber (12) is provided with an IC electrode (19); Each EC chamber (22) includes an EC electrode (29); The manifold (50) a plurality of first pipetting channels (51) arranged in a third array (52); Optionally, a plurality of second pipetting channels (53) arranged in a fourth array (54); the manifold (50) is movable between a first position and a second position relative to the patch clamp plate (100); In a first position, each of the first pipetting channels (51) or each of the second pipetting channels (53) of the manifold (50) is configured to be aligned with one IC inlet (11) of the patch clamp plate (100); A patch clamp system configured such that in the second position, each of the first pipetting channels (51) or each of the second pipetting channels (53) of the manifold (50) is aligned with one EC inlet (21) of the patch clamp plate (100).

2. The manifold (50) comprises a plurality of first pipetting channels (51) arranged in a third array (52) and a plurality of second pipetting channels (53) arranged in a fourth array (54); In the first position, each of the second pipetting channels (53) of the manifold (50) is configured to be aligned with one IC inlet (11) of the patch clamp plate (100); 2. The patch clamp system of claim 1, wherein in the second position, each of the first pipetting channels (51) of the manifold (50) is configured to be aligned with one EC inlet (21) of the patch clamp plate (100).

3. a pipette guide (60) arranged such that a manifold (50) is placed between the pipette guide (60) and the patch clamp plate (100); The pipette guide (60) a plurality of first guide channels (61), each of which is arranged to be aligned with a first pipetting channel (51) of the manifold (50); 2. The patch clamp system of claim 1, optionally comprising a plurality of second guide channels (62), each of which is positioned to align with a second pipetting channel (53) of the manifold (50).

4. 3. The patch clamp system of claim 2, wherein the manifold (50), optionally together with a pipette guide (60), is configured to enable deeper and controlled penetration of a pipette tip into the patch clamp plate (100) via the plurality of second pipette channels (53) than penetration of the same pipette tip into the patch clamp plate (100) via the plurality of first pipette channels (51).

5. The patch clamp plate (10) has a major surface (101), The major surface (101) comprises a plurality of intracellular (IC) inlets (11) arranged in a first array (10) and a plurality of extracellular (EC) inlets (21) arranged in a second array (20); 2. The patch clamp system of claim 1, wherein the manifold (50) is suitably positioned adjacent to a major surface (101) of the patch clamp plate (100) and is movable relative to the patch clamp plate (100) at least in a plane parallel to the major surface (101).

6. an actuator (90) arranged to drive the manifold (50) relative to the patch clamp plate (10) between the first and second positions; Preferably, the actuation is performed at least in a plane parallel to the main surface (101), More preferably, the actuator (90) is a linear actuator.

7. The actuator (90) is also arranged to drive the manifold (50) in a second plane; The second plane is perpendicular to the main surface (101); 7. The patch clamp system of claim 6, wherein the actuator is preferably arranged to clamp the first surface of the manifold relative to the major surface of the patch clamp plate at the first and second positions.

8. 2. The patch clamp system of claim 1, wherein the patch clamp plate further comprises a plurality of IC pressure relief outlets arranged in a sixth array and a plurality of EC pressure relief outlets arranged in a seventh array, each IC pressure relief outlet being fluidly connected to an IC chamber and each EC pressure relief outlet being fluidly connected to an EC chamber.

9. The manifold (50) further comprises one or more IC pressure channels (55) arranged in a fifth array (56); 2. The patch clamp system of claim 1, wherein the IC pressure channels (55) are arranged such that, in the second position, each of the IC pressure channels (55) of the manifold (50) is configured to be aligned with a respective IC inlet (11) of the first array (10).

10. The manifold (50) further comprises one or more EC pressure channels; 2. The patch clamp system of claim 1, wherein the EC pressure channels are arranged such that, in the second position, the EC pressure channels of the manifold are configured to align with each EC pressure relief outlet.

11. further comprising at least one pressure control device (70); 10. The patch clamp system of claim 9, wherein the at least one pressure control device (70) is fluidly connected to each of the IC pressure channels (55) and / or each of the EC pressure channels in the manifold (50) and is configured to increase or decrease the pressure in each of the IC pressure channels (55) and / or each of the EC pressure channels of the manifold (50).

12. A process for priming a patch clamp system according to any one of claims 1 to 11, comprising: - providing a patch clamp system according to any one of claims 1 to 11; With the manifold (50) in the first position, pipetting IC fluid into each intracellular (IC) chamber (12) of the patch clamp plate (100) through the IC inlet (11) using a second pipetting channel (53) in the manifold as a pipette guide; - moving the manifold (50) to a second position; Pressurizing each IC chamber via one or more IC pressure channels in the manifold; pipetting EC fluid into each extracellular (EC) chamber (22) of the patch clamp plate (100) through the EC inlet (21) using a first pipetting channel (51) in the manifold as a pipette guide; and pressurizing each EC chamber via one or more EC pressure channels in the manifold.

13. A process for trapping one or more biological cells in a patch clamp system according to any one of claims 1 to 11, comprising:

13. The priming process of claim 12, further comprising: With the manifold (50) in the second position, pipetting biological cells (C) into each extracellular (EC) chamber (22) of the patch clamp plate (100) through the EC inlet (21) using a first pipetting channel (51) in the manifold as a pipette guide; - applying negative pressure through the patch hole (26) to trap biological cells at said patch hole (26).

14. A process for determining and / or monitoring electrophysiological properties of ion channels in biological cells, in a patch clamp system according to any one of claims 1 to 11, comprising: - providing a patch clamp system according to any one of claims 1 to 11; - priming the patch clamp system according to the process of claim 12; - capturing one or more biological cells in said one or more patch holes (26) according to the process of claim 13; - performing electrophysiological measurements on said biological cells.

15. A patch clamp device (200), said device (200) comprising a patch clamp system according to any one of claims 1 to 11, a computer system having software, said computer system and associated software comprising: o controlling the pressure control device (70); o controlling the actuator (90); o controlling one or more patch clamp experimental parameters; o measuring electrical signals generated by cells trapped in a patch clamp system via the IC electrodes (19) and the EC electrodes (29); The device (200) further comprises: a user interface for communicating with a user of the device, the user interface being in electronic communication with the computer system; Optionally, a patch clamp apparatus (200) comprising a housing (201) in which said patch clamp system and said computer system are placed.

Images