Patent Application
20250076278
The invention relates to a patch-clamp apparatus comprising a patch-clamp plate manipulator configured to receive a patch-clamp plate comprising a plurality of patch-clamp sites. The patch-clamp apparatus comprises a computerised controller. The invention further relates to a method of carrying out a patch-clamp procedure.
Ion channels are membrane proteins in cells of living organisms. A flow of ions into and out of the cells is controlled by the membrane, and the ion channels are important targets for a variety of drugs.
Ion channels are commonly studied with a technique called patch-clamping. The cells are applied to a patch-clamp plate comprising a plurality of patch-clamp sites.
The on-cell, whole-cell, or perforated patch configurations of patch-clamp are widely accepted as providing the best methods of measuring ion channel activity for drug screening. In these methods, ion currents flowing through ion channels are measured directly and with high resolution by sensitive current amplifiers.
A protocol specifies a pressure, a potential, or a current which is applied to the patch-clamp site, and a current, a capacitance, or a cell potential etc. is subsequently measured across the ion channel.
Users need efficient methods to investigate ion channels. While a high throughput in the screening process is important, flexibility and freedom for the user to dynamically interact with the investigation process is highly desired.
The general idea of electrically insulating a patch of membrane and studying the ion channels in that patch under voltage-clamp conditions is outlined in Neher, Sakmann, and Steinback (1978) “The Extracellular Patch-clamp, A Method For Resolving Currents Through Individual Open Channels In Biological Membranes”, Pflüger Arch. 375; 219-278. Recent developments in patch-clamp methodology have seen the introduction of planar substrates (e.g a silicon chip) in place of conventional glass micro pipette (for example, see WO 01/25769 and Mayer, 2000). Additional background art includes U.S. Pat. No. 8,268,260, which is hereby incorporated by reference.
Unfortunately, current patch-clamp methods and devices suffer from shortcomings. Particularly, existing methods are either manual methods or fully automatic methods. The manual methods require highly skilled operators performing time consuming testing, and throughput is typically insufficient for industrial testing. The automatic methods are carried out by large and expensive machines with very little user interaction once an experimental protocol has started. While offering a high throughput, they do not offer the opportunity to dynamically amend ongoing experiments, and each experiment must be planned from start to end before it is initiated.
It is an object of embodiments of the invention to improve patch-clamping and particularly to improve the balance between throughput and dynamic interaction during testing. It is a further object to effectively bridge a gap between manual and automated measurements while reducing operator error and to allow a more robust, reproducible measurement known from automated measurements while retaining the flexibility of manual patch experiments demanded by expert users.
For this and other purposes, the invention, in a first aspect, provides a semi-automatic apparatus wherein the controller is configured to:
The controller initiates the process steps assigned to automatic execution by dispatching the commands to the manipulator and initiates the manual process steps assigned to manual execution by dispatching the instructions to the user.
Since the controller has the patch-clamp experiment as a defined procedure with a plurality of steps, and each step is assigned to either manual or automatic execution, the controller may change between manual and automatic processing during a patch-clamp procedure. This provides improved throughput during automatic processing and preserves the ability to dynamically interact during the experiment.
When used herein, “semi-automated” means that the apparatus is configured to allow certain process steps to be carried out manually while allowing automatic execution of other process steps.
When used herein “patch-clamp procedure” means a selection of process steps and a specific order they are carried out.
When used herein “process steps” means single activities included in the patch-clamp procedure and they could be defined e.g. in a protocol.
When used herein “protocol” means a pre-specified electrical signal, pressure signal, optical signal, or combinations thereof which can be applied to the patch-clamp site by the manipulator.
The patch-clamp plate manipulator is configured to carry out all necessary handling of the patch-clamp plate. The manipulator comprises mechanical components including inlets facilitating supply of solutions to the patch-clamp sites and for controlling pressure at the patch-clamp sites. Additionally, the manipulator comprises electronic components for carrying out a procedure where an electrical and/or a pressure signal and/or an optical signal is applied to the patch-clamp site and an electrical signal is received from the patch-clamp site.
The computerised controller is configured with a user interface for communication with the user and with a data interface to the manipulator such that the controller can control the manipulator and thus the automatic process steps. Additionally, the controller may have other interfaces, e.g. for communicating data with external computers.
The patch-clamp procedure comprises a plurality of pre-defined process steps. These process steps are defined in the memory of the controller, and they include manual process steps to be executed manually by the user and automatic process steps for automatic execution by the manipulator. The controller is programmed to provide instructions both relative to the manual process steps and relative to the automatic process steps.
The instructions for the user are typically provided on a computer screen, e.g. in written form, and typically with graphical interaction illustrating e.g. where to add solutions etc. The interface may also feed results back to the user during execution of the procedure and thereby allow the user to amend subsequent steps in view of the results obtained by previous steps. The controller may be configured to communicate with the user not only by the screen, but also by audio signals or combinations of optical signals and audible signals.
The controller may particularly comprise a plurality of patch-clamp procedures. The user may select a predefined patch-clamp procedure from a procedure library and thereby have several predefined process steps loaded into the controller and ready to be carried out.
The controller may also allow the user to define new patch-clamp procedures. These may be defined e.g. via the computer screen or they may be uploaded from another computer system.
For defining new patch-clamp procedures, the controller may comprise a plurality of predefined process steps. Each process step may be predefined either as an automatic process step or a manual process step.
The controller may comprise a plurality of patch-clamp protocols. Each protocol defines a sequence of signals applied automatically by the manipulator to the patch-clamp sites. The protocols may be included in a protocol library from which the user can select a protocol for a specific process step. At least one of the protocols may specify at least one of a pressure, a potential, or a current applied by the manipulator to the patch-clamp site.
Additionally, the controller may allow the user to define and optionally to store new protocols when needed. These may be defined e.g. via the computer screen or they may be uploaded from another computer system.
The protocols may, in addition to pressure and electrical signal values, specify an optic signal to flash the cell, e.g. by light of different wave-lengths, e.g. ultra-violet light e.g. to provide light-evoked currents recorded under voltage clamp.
The controller may comprise measurement steps defining how readings are made automatically by the manipulator at the patch-clamp sites. At least one of the measurement steps may specify reading of an electrical signal or an optical signal from the patch-clamp site.
The controller may also allow the user to define new measurement steps. These may be defined e.g. via the computer screen or they may be uploaded from another computer system.
The controller may allow the user to select predefined protocols and/or predefined measurement steps for a patch-clamp procedure, and particularly to allow such selection while the patch-clamp procedure is carried out. This allows the user to change the patch-clamp procedure depending on results obtained during the procedure.
The controller may be configured to assign the manual process steps to a time critical group of manual process steps or to a non-time critical group of manual process steps.
The instructions for a user to carry out steps assigned to manual execution may be provided with a timer countdown triggering automatic shifting to a subsequent manual process step or automatic process step execution when the manual process step is assigned in the time critical group of manual process steps. This allows the controller to determine the timing between two subsequent process steps and thereby ensures that certain timing parameters are observed.
A user triggered acknowledgement may trigger a subsequent manual process step or automatic process step execution when the manual process step is assigned in the non-time critical group of manual process steps. This allows the user to determine the timing when the timing is less critical for the experiment being carried out. The user triggered acknowledgement could be pushing of a button on the touch screen, or voice activated acknowledgement, e.g. by the user giving a “next” command.
The controller may be configured to allow the user to re-assign a manual process step to an automatic process step, or to re-assign an automatic process step to a manual process step. This may e.g. by done during the execution of a patch-clamp procedure.
The controller may be configured with a user interface allowing the user to store a patch-clamp procedure after having amended the patch-clamp procedure, and optionally to add meta-data related to a patch-clamp procedure. The controller may define a data file comprising the meta data with data related to the patch-clamp procedure, and it may allow such meta-data to be exported to another computer system. The user interface may allow the user to add the meta data individually to selected process steps or to the patch-clamp procedure as such. In one example, the user may e.g. make a comment to a result obtained in a measurement step, or the user may make a comment about the entire procedure.
Additionally, or alternatively, the controller may automatically add meta data related to the patch-clamp procedure and/or to individual process steps, e.g. data and time stamps, or results obtained in measurement steps.
The controller may be configured to communicate to the user which process step is being executed, e.g. by visualising graphically on the screen the current step, the previous step and the next step or by visualising that the current step is step nr. x out of a total number of y steps.
The controller may be configured to allow the user to skip a process step and jump to a subsequent process step and to communicate results of a process step to the user before initiating a subsequent process step.
The controller may comprise a graphical user interface and the instructions for a user to carry out manual process steps may comprise graphical illustrations related to the patch-clamp plate or the plurality of patch-clamp sites, e.g. illustrating specifically in which inlet to add a particular solution etc.
The controller comprises a predefined sequence in which the process steps are executed. The sequence may e.g. determine that one process step is carried out after another process step and before yet another process step. The sequence may also determine that two process steps are carried out simultaneously after another process step and before yet another process step. The controller may comprise a user interface allowing the user to change the sequence, e.g. by keying in a different sequence, or by graphically drag-and-dropping a step to a different location in a sequence of steps. A change in the sequence may be added either automatically or manually to said meta data.
In a second aspect, the invention provides a method of carrying out a patch-clamp procedure, the method comprising predefining the procedure in the form of a plurality of manual process steps and a plurality of automatic process steps in a computer system and allowing the computer system to trigger execution of the automatic process steps by electronically controlling an actuator and to trigger the manual process steps by instructing a user.
The method may include any step implicit in view of the apparatus according to the first aspect of the invention.
Further details of the technology are provided in the enclosed dependent claims, figures, and examples.
The technology is illustrated by means of the following schematic illustrations, in which:
The patch-clamp technique is used to study ionic currents in individual isolated living cells, tissue sections, or patches of cell membrane. The technique is inter alia used in studies of neurons, cardiomyocytes, muscle fibres, and pancreatic beta cells.
Different patch-clamping techniques are used. In voltage clamp technique, the voltage across the cell membrane is controlled, and the resulting currents are recorded. In current clamp technique, the current passing across the membrane is controlled and the resulting changes in voltage are recorded.
The patch-clamp plate is of a kind used in automated patch-clamp (APC) measurements for high-throughput recordings of ion channel currents in living cells. Such patch-clamp plates are assembled from several components with a range of properties that are advantageous for APC measurements.
The patch-clamp plate is typically “single-use”. Accordingly, they are easily replaced by removing a used, plate from the slot 101, and inserting a new single-use plate in the slot. In this manner, contamination between experiments can be reduced or eliminated.
The patch-clamp plate comprises a plurality of intracellular (IC) inlets arranged in at least one array. Each IC inlet is in fluid connection with an IC chamber. The patch-clamp plate also comprises a plurality of extracellular (EC) inlets arranged in another array. Each EC inlet is in fluid connection with an EC chamber.
The patch-clamp plate also comprises a patch-clamp chip arranged between each EC chamber and each IC chamber, each patch-clamp chip comprising one or more patch holes. The at least one patch hole extends through the patch-clamp chip and provides a fluid connection between EC and said IC chambers. The biological cells to be tested are captured on the patch hole during patch-clamp experiments.
Each IC chamber of the patch-clamp plate comprises an IC electrode; and each EC chamber comprises an EC electrode.
The patch-clamp plate may additionally comprise a cover which shields the testing environment and provides inlets for liquid access to the experiment site. A flexible gasket may also be present, to form the liquid- and electrical seal between the EC and IC chambers.
At the experiment site, a tight, giga-ohm (GQ) seal is established between the cell membrane and patch hole using physiological solutions. This allows a user to avoid “seal-enhancing” ions, such as fluoride, which are often required in APC experiments and have been reported to modulate ion channel function
The patch-clamp plate may contain multiple measurement sites depending on the required throughput. Each “measurement site” comprises the IC chamber, EC chamber and at least one patch-clamp chip, with associated EC/IC inlets and outlets.
The microfluidic channel on the extracellular side allows rapid liquid exchange with multiple solution additions to the same cell during an experiment. The solution consumption is significantly reduced as solution exchange can be achieved with less than 10 μL of solution
The apparatus comprises an array of inlets 102 for liquid addition e.g. by use of manual pipetting. Any number of inlets may be provided. In the disclosed apparatus, there is an array of 8 inlets. The array contains 8 inlets configured for receiving 8 separate pipets.
The apparatus comprises a patch-clamp plate manipulator receiving the patch-clamp plate and configured to carry out automatic procedures. The manipulator comprises mechanically moving components for bringing the 8 separate inlets in communication with the EC and/or IC chambers of the plate. Subsequently, the 8 different solutions can be treated individually.
The manipulator also comprises required electronic components for applying a signal to the patch-clamp site and for reading a signal from the patch-clamp site. The applied signal includes an electrical signal, a pressure signal, and optionally, an optic signal, e.g. a flash of light of a certain wave-length.
The apparatus comprises a connection for pressurised gas, e.g. air, vacuum, and optionally a connector for water or for communication of other consumables for the experiment.
The apparatus comprises a computerised controller configured with a user interface in the form of a computer screen 103, in this case a touch screen allowing two-way communication between the controller and the user.
The controller is implemented in a CPU with memory and computer executable code for enabling various functions which will be described in further details below. The illustrated apparatus comprises a dedicated controller specifically made for carrying out the patch-clamp procedure. The controller could, alternatively, be constituted at least partly by a standard computer system, e.g. a PC. The controller comprises a data interface for communication of data externally, e.g. for exporting results and for importing patch-clamp procedures, process steps, and/or protocols.
Those skilled in the art will appreciate that the functions of the patch clamp device may be implemented using standard hardware circuits, using software programs and data in conjunction with a suitably programmed digital microprocessor or general-purpose computer, and/or using applications specific integrated circuitry, and/or using one or more digital signal processors. Software program instructions and data may be stored on a non-transitory, computer-readable storage medium, and when the instructions are executed by a computer or other suitable processor control, the computer or processor performs the functions associated with those instructions.
Devices known in the art are made for fully automatic procedures where the entire procedure is prespecified in the memory of the controller. This is efficient but results in reduced flexibility and very little interaction with the user. The controller of the apparatus illustrated in
Some of the process steps are for manual process execution by the user and some of the process steps are to be carried out automatically by the apparatus.
The controller provides instructions for the user relative to the manual process steps, and it controls the manipulator relative to automatic execution of the automatic process steps.
The patch-clamp procedure comprises a plurality of process steps. In this implementation of the invention, the process steps are grouped in four different groups of process steps.
A first group of process steps is referred to as “set-up” or simply group S and contains 8 process steps in which an experiment is being setup. The number of these process steps begin with an S, i.e. S1-S8.
A second group of process steps is referred to as “Priming of patch-clamp plate” or simply group P and contains 9 process steps in which the patch-clamp plate is being primed. The number of these process steps begin with a P, i.e. P1-P9.
The priming process may include a priming protocol by which complete evacuation of air from the microfluidic channels before the beginning of the experiment is carried out.
Cells are applied via an extracellular channel and positioned at the patch hole(s) by low, negative pressure across the patch hole(s). When properly sealed, a brief suction pulse brings the cell into the whole-cell configuration, where it is held in place by low, negative pressure throughout subsequent patch clamp experiments. The extracellular solution can be exchanged throughout the experiment allowing precise addition and washout of solutions.
A third group of process steps is referred to as “Cell positioning and Whole Cell (WC) formation” or simply group C and contains 5 process steps in which the cell is positioned, and WC is formed. The number of these process steps begin with a C, i.e. C1-C5.
A fourth group of process steps is an optional, free definable set of process steps. This could be a proprietary standard used by a certain user. Herein, this is referred to as “E-phys experiment” or simply group E and contains 12 process steps in which the E-phys experiment is carried out. The number of these process steps begin with an E, i.e. E1-E12.
The process steps are outlined below:
Each step includes an indication [A] for automatic or [M] for manual. This is a definition comprised in the controller and distinguishing manual process steps from automatic process steps. During execution of a patch-clamp procedure, the identification of automatic and manual process steps allows the controller to execute the patch-clamp procedure by dispatching the commands to the manipulator for execution of the automatic process steps and by dispatching the instructions to the user for manual execution of the manual process steps.
In Step S1, the corresponding instruction dispatched by the controller via the touch screen is “please insert a new patch-clamp plate”.
In Step S2, the corresponding command to the manipulator is to scan the barcode on the patch-clamp plate. This is an electronic command submitted to a bar code reader included in the patch-clamp plate manipulator, and in response to receiving this instruction, the bar code reader reads a bar code on the patch-clamp plate and returns a data string representing the read value.
In Step S3, the corresponding instruction dispatched by the controller via the touch screen is “please select a number of available sites”
In Step S4, the corresponding instruction dispatched by the controller via the touch screen is “please add meta-data”, or the instructions are guiding the user to add specific data. In an example, the user may benefit from pull-down menus where a patch-clamp procedure ID, date, user-ID and other kinds of predefined meta-data can be selected and associated with the patch-clamp procedure which is being initiated.
In Step S5, the corresponding instruction dispatched by the controller via the touch screen is “please select solutions” and the user is guided by pull-down menus where different solutions and concentrations can be selected and associated with the patch-clamp procedure which is being initiated.
In Step S6, the corresponding instruction dispatched by the controller via the touch screen is “please select a protocol” and the user is guided by pull-down menus where different protocols from a library of protocols can be selected and associated with the patch-clamp procedure which is being initiated.
In Step S7, the corresponding instruction dispatched by the controller via the touch screen is “please select a sequence” and the user is guided by a graphical user interface illustrating a pre-defined sequence of steps and protocols for the patch-clamp procedure, and the user can change the predefined sequence.
In Step S8, the corresponding instruction dispatched by the controller via the touch screen is “please start the patch-clamp procedure by pressing start” and the user has the option to wait until being ready. At this point, additional menus may allow the user to abort the procedure, and/or to store the procedure for execution at a later point in time.
Additionally, the user can cancel steps, or insert additional steps between existing steps.
In
The present invention has been described with reference to several embodiments and figures. However, the skilled person can select and combine various embodiments within the scope of the invention, which is defined by the appended claims. All documents referenced herein are incorporated by reference.
The controller comprises protocols each defining a sequence of signals applied automatically by the manipulator to the patch-clamp sites. Additionally, the controller comprises measurement steps defining readings made automatically by the manipulator at the patch claim sites.
The user interface illustrated in
The user interface illustrated in
All user interfaces allowing the user to define or amend a patch-clamp procedure, a process step, a protocol, or a measurement step is complemented with a save-selection button by which the user can instruct the controller to store the amendment either by overwriting the previous patch-clamp procedure, process step, protocol, or measurement step or by creating a new patch-clamp procedure, process step, protocol, or measurement step.
By a simple skip command, the user can skip a process step by the delete button 84 and jump to a subsequent process step.
| Number | Date | Country | Kind |
|---|---|---|---|
| 22150580.3 | Jan 2022 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2023/050288 | 1/9/2023 | WO |
