PatchXpress 7000A

Automated Parallel Patch-Clamp System

 
PatchXpress® 7000A Automated Parallel Patch-Clamp System is a tool that will revolutionize ion channel drug discovery. It is the realization of nearly every patch clamper's dream. The PatchXpress directly records ion channel function in a patch clamp screening assay and obtains thousands of data points per day, instead of the one to ten data points standard with conventional patch clamp techniques.

The SealChip™ electrodes give the PatchXpress system its edge. These planar electrodes turn conventional patch clamp electrodes upside down, and flatten them out, making them amenable to a multiwell format for testing multiple cells. The PatchXpress is completed by robust, accurate, and highly configurable amplification and fluidic systems.

Seriously Tap into Ion Channel Targets for the First Time

Because they play important roles in cell physiology such as excitability and gene expression, ion channels are ideal drug discovery targets. Despite their theoretical suitedness as targets, ion channels have been a relatively untapped resource in the world of drug discovery. There have been no means to directly measure ion channel activity in the context of a high throughput patch clamp assay, so researchers have used other, less direct techniques for screening. The PatchXpress 7000A changes all this, opening up a huge pool of highly "drugable" ion channel targets.

The key to direct measurements with the PatchXpress is voltage control. Other methods (e.g., fluorescence based methods) do not control membrane voltage, so the end result is indirect measurement of ion channel activity. This leads to costly false negatives and false positives. In contrast, patch clamping allows you to control the voltage in a single cell and directly measure the current flowing through its ion channels. The high information content in such a measurement gives more than just a "yes/no" response for each compound tested. For example, you can use voltage control to stimulate cells at particular frequencies, generating use-dependence data of ion channel function to be exploited in the design of smart drugs. For PatchXpress, frequency modulation is a breeze.

PatchXpress Reduces the Cost of Electrophysiology

At the same time it increases throughput, PatchXpress dramatically lowers the cost per data point (where one data point is produced for each compound tested). At the moment, most ion channel drug discovery groups employ highly trained electrophysiologists to run patch clamp rigs. Without automation throughput is low-even the best patch clampers struggle to get 50 data points a week. With the PatchXpress, technicians will be able to easily measure up to 1200 data points per week per system. This represents more than an order of magnitude cost reduction, a savings you cannot afford to miss out on if you are serious about ion channel drug discovery.

SealChip16:
A Key to Increasing Throughput

Planar patch clamp with SealChip16 is a key to increasing throughput in ion channel drug discovery. Its open-well format provides access to automation and parallel processing. No more cumbersome glass patch clamp pipettes! The PatchXpress system automatically places a cell in each well of the 16-channel electrode array for patch clamping. Gigaseals and whole cell recordings are obtained with independent pressure control and data acquisition for each channel. Parallel patch clamping of sixteen cells per chip allows testing up to 240 compounds in one eight-hour day - a feat impossible with a conventional patch clamp.

Press the Play button to start the PatchXpress 7000A animation.
Turn on your speakers to hear the narration. A full-screen version is
also available on the Literature & Downloads link in the right menu.

Aviva's planar electrodes have a very high success rate in forming gigaseals with mammalian tissue culture cells. Most cells proceed to whole cell configuration and last for at least 15 minutes, with acceptable values of membrane resistance (Rm), access resistance (Ra) and holding current throughout the recording period. The robust recordings afforded by SealChip16 electrodes easily accommodate a typical 3-phase test of a single compound, incorporating baseline recording, application of the test compound, and then another baseline measurement following washout to determine whether or not the effect of the compound is reversible.

PatchXpress SealChip16

The SealChip16 electrode consists of 16 planar patch clamp holes of about 1 µm diameter each, aligned on a single strip 4.5 mm apart. Each planar electrode acts independently to record currents from mammalian tissue culture cells. A well above each hole holds 50 µl to 100 µl of external solution, while a much smaller compartment underneath each hole holds internal solution. All together, each SealChip16 electrode looks like a single column in a 384-well plate. As with a conventional patch clamp, the SealChip16 electrodes are disposed after each use because debris from a patch clamped cell prevents subsequent gigaseal formation.

For a closer look at a one-channel planar patch clamp experiment with RBL cells, download and view this Aviva video [AVI].

High Quality Automated Patch Clamp

With the PatchXpress 7000A each channel is independently controlled for optimal recording quality. For example, independent pressure control means better gigaseal formation on each channel, because the time to form the gigaseal can vary so much from cell to cell. Independent channels mean recordings with less noise. Independent pressure control also translates to lower whole cell access resistance values, with finer control of this important membrane parameter.

Automated pressure control for optimal membrane parameters

During a patch clamp experiment, the seasoned electrophysiologist keeps a close eye on important membrane parameters including Rm, Ra and holding current. Changes in these membrane parameters signal the patch clamper that something could be wrong with recording quality. In the worst case, changes in current amplitude occur that relate to the changes in the membrane parameter values and not to changes in the biology of the cell. The smart patch clamper will use pressure control or voltage "zaps" to optimize these parameters and ensure reliable measurements during the life of the recording.

The membrane resistance value, Rm, relates to the initial gigaseal achieved and the input resistance of the cell (in general, larger cells have a lower input resistance because they have more channels open at rest or "leak" channels). Independent and automated pressure control in the PatchXpress affords tight gigaseals at the onset of each experiment. Tight gigaseals are critical for low noise, stable, and long-lasting whole cell recordings.

The access resistance value, Ra, relates to the initial electrode resistance plus the internal resistance of the cell. For example, if internal organelles clog the opening of the planar patch clamp electrode, the Ra value will increase. If the Ra value is too high and the measured current amplitude is greater than 1 nA, a significant voltage error occurs because of Ohm's Law (Voltage = Resistance * Current). For example, if the Ra value reaches a point of 20 MΩ during a recording and the current amplitude is 1 nA the resulting voltage error would be 20 mV! One can easily see how this could lead to a false positive or false negative during a compound screening experiment. The PatchXpress system uses automated pressure control to its advantage to maintain a relatively constant Ra value throughout the recording period.

Fluidics for recording ligand-gated and voltage-gated ion channels

Screenshots of the PatchXpress animation

PatchXpress includes the following fluidics: Disposable pipettes for placing cells in the SealChip16 electrode wells. Disposable pipettes for applying test compounds. Washout probes for automatic washout of test compounds with buffer solutions.

Compound application can be timed precisely to occur at just the right moment in the sequence of the experiment. Disposable pipette tips ensure no cross-contamination between drug applications. The fluidics system can be configured for single applications or for cumulative dose-response applications.

As shown in Figure 1, the application of test compound is rapid enough for both ligand-gated and voltage-gated ion channel studies. The figure shows results from experiments done with RBL cells that endogenously express inward rectifying potassium channels. The holding potential was -80 mV and a high potassium external solution was added to change the holding current. The time to 90% exchange is in the order of hundreds of milliseconds and is indicated by the time taken to reach the new holding level. Washout is achieved with a separate buffer line and reaches its peak and plateaus in about 100 ms (see Figure 1).

Figure 1.

Screening Both Voltage and Ligand-gated Channels is a Breeze

It is reasonable to expect an automated patch clamp system to record currents from both voltage and ligand-gated channels. The unique PatchXpress planar electrodes and fluidics system afford quality patch-clamp recordings of both ion channel types.

Figure 2 shows an example of voltage-activated currents through Kv1.1 potassium channels expressed in CHO cells (from Dr. William Thornhill, Fordham University). Panel A (blue traces) shows uncorrected or raw Kv1.1 data in response to a family of voltage steps from -60 to +60 mV in increments of 10 mV. Panel B (red traces) shows data corrected with software controlled linear leak and capacitance correction (P/4 routine). As shown in the I-V curves in Panel C, the recordings are stable and similar with or without the P/4 routine.

Figure 2. Kv1.1 potassium channel currents with (A) raw data and (B) P/4 linear leak
and capacitance subtracted data. Panel C shows the I-V relationship from both
experiments. Data courtesy of Drs. Jia Xu and Antonio Guia (AVIVA Biosciences).

Figure 3 shows an example of desensitizing ligand activated currents in response to direct application of 100 µM and 1 mM acetylcholine (Ach) in the same cell patch-clamped with a planar electrode. Automated whole cell patch clamp recordings (holding potential -80 mV) started with a gigaseal (> 1 GΩ). Transition to whole cell mode was under also under automated control using a software controlled amplifier (MultiClamp 700A) and pressure controller. Inward currents were recorded in response to ACh applied using PatchXpress robotic fluidics system. The contact time with ACh is indicated by the length of the bar. The well was washed out using perfusion pipes sited in close proximity to the base of the recording well in order to achieve a rapid wash out.

Figure 3. Ligand activated currents in response to
direct application of 100 µM and 1 mM Acetylcholine.

PatchXpress Software Monitors Patch Clamp Experiments:
A Virtual Electrophysiologist

The central nervous system of the PatchXpress is its software. Beside the straightforward control of the PatchXpress hardware systems, the PatchXpress program makes critical decisions while an experiment is actually running-just like a seasoned patch clamper—in order to keep the experiment on track.

The PatchXpress software makes experimental setup intuitive and user-friendly. It is even possible for pCLAMP users to import their familiar episodic parameter files.

During experiments, the PatchXpress software masterfully controls all of the robotic instrumentation while seamlessly displaying useful information to the user about the status of the patch clamp experiments on each of the 16 channels.

Following is a sequence of events during a typical experiment under the control of the PatchXpress software:

load SealChip16 electrode load electrodes with internal solution apply cells apply just the right amount of pressure to obtain a gigaseal apply pressure until the whole cell configuration is achieved start voltage step protocol and record baseline currents monitor membrane parameters throughout apply test compound start voltage step protocol and record currents washout test compound start voltage step protocol and record currents create quality control log file with detail information about the experiment (see example figure)

The step "monitor membrane parameters throughout" is highlighted because it deserves clarification. Before each screen, the user predefines the acceptable criteria for a successful recording. For example, the tolerance for Ra and Rm may be 15 MΩ and 500 MΩ, respectively. In this case, if Ra goes above 15 MΩ and/or Rm dips below 500 MΩ the PatchXpress software needs to act quickly. The first step would be to apply suction (negative pressure) in an effort to clear any obstruction (i.e., internal organelle) that could be responsible for the increase in Ra. In the best-case scenario, this negative pressure would also improve the seal and cause Rm to go back (or remain) above 500 MΩ. In the worst-case scenario, the software would make a note that the criteria for a successful recording have not been met and this would be apparent at the user interface. Importantly, in the latter case, valuable test compound would not be applied to this cell. The PatchXpress software and pipetting robotics work together to apply test compounds only to cells that meet the criteria for a successful recording.

Data output from the PatchXpress will be in the form of standard text files and Axon Binary Files (ABF). These ABF files can be imported to DataXpress for automated analysis or to Clampfit for detailed manual analysis. Some on-line analysis, such as peak current amplitudes, is also available during PatchXpress experiments (and exported as text files).

DataXpress is a database application designed for data management and automated, macro-driven batch analysis. DataXpress will automatically generate IV and dose-response graphs. On-line data from PatchXpress experiments can be ported directly to DataXpress where hits and misses in a drug screen are displayed.

Figure 4.  An example of the membrane parameters recorded during a planar patch clamp experiment that shows the quality of the recording. In this example, Ra remained below 15 MΩ and Rm stayed above 500 MΩ for longer than 15 minutes. Holding current and whole-cell capacitance values are also displayed.

Two Decades of Experience

The PatchXpress 7000A emerges from years of Axon's experience as the world leader in electrophysiology hardware and software. For the past two decades, Axon provided tools for electrophysiologists performing cutting edge ion channel research in both academic and pharmaceutical company laboratories. The PatchXpress 7000A builds on these core technologies and includes the revolutionary SealChip technology that allows recordings from many cells in parallel for increased throughput for ion channel screening.

MultiClamp Amplifiers: the Silent Workhorses

Tucked away in the PatchXpress 7000A are eight MultiClamp 700A amplifiers, each connected with two CV-7A headstages. These computer-controlled patch clamp amplifiers have been proven in the field for over two years and are ideal for automated patch clamping because there are no knobs and buttons. The PatchXpress software controls the settings on each amplifier channel, allowing the user to make choices such as the following:

• current clamp vs. voltage clamp
• 50 MΩ, 500 MΩ, 5 GΩ, or 50 GΩ feedback resistors for voltage clamp
• 60 lowpass Bessel filter settings

By incorporating MultiClamp amplifiers, the PatchXpress 7000A gives you the flexibility to perform detailed research mode experiments or higher throughput screening experiments: both with the same high fidelity and data quality Axon customers have enjoyed over the years.

Patch Clamp Data Acquisition: Our Specialty

Ask any electrophysiologist what the industry standard is for electrophysiology software, and the answer will undoubtedly be pCLAMP. Developing flexible and user-friendly electrophysiology software is a daunting task. We have achieved this with pCLAMP, with nearly two-decades of experience behind each new update version. This same expertise went into developing PatchXpress software.

For conventional patch clamp applications, the Digidata 1322A (see figure) facilitates communication from the computer to the amplifiers (Digital-to-Analog or "D to A" conversion) as well as from the amplifiers to the computer (Analog-to-Digital or "A to D" conversion). The PatchXpress incorporates a modified Digidata that has 16 Analog Inputs and Outputs (the Digidata 1322A has only two analog outputs).

The PatchXpress 7000A also builds upon experience gained by the OpusXpress 6000A, the world's first parallel electrophysiology recording system. In developing the OpusXpress, Axon gained valuable experience in fluidics and electrophysiology acquisition software for ion channel screening applications. The following quote from OpusXpress user Dr. Roger Papke (Univ. Florida), describes the advantage of using high-quality, automated voltage clamp systems:

"With the PatchXpress and OpusXpress product lines, Axon has brought the quality of academic research to the high throughput needs of drug research and development. At the same time, it offers an unprecedented level of potential productivity to the academic researcher who has access to these systems. Our laboratory was fortunate to participate in the OpusXpress beta test program. In the course of a month we completed a project that otherwise might have taken as long as two years."

Features and Specifications

The PatchXpress is an automated high-throughput electrophysiology workstation.

• Automated parallel patch-clamp recording of multiple mammalian cells simultaneously
  • Gigaseals (cell-attached resistance > 1 GΩ) to start each recording
  • True whole-cell recordings
  • Low access resistance (Ra) values achieved by 16 independent pressure controllers
  • 16 independent patch clamp amplifiers
  • High-fidelity 16-channel input/output digitizer based on the proven technology of the Axon Instruments Digidata 1322A
  • Dimensions: 58" wide x 30" deep x 66" tall (plus 24" monitor attached to the side)
• High speed whole-cell voltage and current clamp capability
  • 8 MultiClamps with 16 CV-7A headstages (a proven technology)
  • 4 different feedback resistors in voltage clamp mode allow versatile high fidelity recordings whether currents are in the pA or nA range
  • Current clamp is true and fast because the CV-7A headstages include voltage-follower circuitry

• Single use planar patch clamp electrode substrate (16-channel)
  • Aviva chips with very high whole-cell recording success rate (gigaseal, 15 minute duration, Ra < 15 MΩ (uncorrected) and Rm > 200 MΩ)
  • Planar format allows compound addition from the top
  • Disposable electrode/chamber means no cross-contamination

• Integrated and intelligent fluidics facilitate drug application during recordings
  • Test compounds are added only to cells with successful recordings to eliminate waste of precious materials
  • Washout of chamber supported
  • Rapid solution exchange
  • Small volumes of test compounds (~ 50 µl)
  • Disposable tips eliminate cross-contamination
  • Cumulative dose-response experiments supported

• Integrated acquisition and analysis software
  • 16 scope windows for live views of recordings in all 16 channels
  • "Chart-recorder" windows for viewing on-line statistics, such as peak amplitude over time
  • Wide-screen monitor for easy viewing of 16 scope windows and "chart-recorder" windows
  • Text file output showing hits based on user criteria
  • Analysis software for data management, quick I-V graphs, and quick dose-response curves

Ordering Information

PatchXpress 7000A

• Integrated hardware and software package comes complete with all hardware, electronics, and software required for full operation, including a computer fully configured with PatchXpress software.