Science Pool

Automated Patched Clamp Electrophysiology: Intracellular Exchange Assay

“The intracellular exchange assay design enables studying the cumulative effect of modulators from the intracellular face of ion channels.” 


Intracellular binding sites of ion channels are targets for many drugs (example: anesthetics targeting voltage gated sodium channels). Pharmacological manipulation of the intracellular (IC) environment is as critical as the extracellular (EC) environment.  

In manual patch clamp, once the gigaseal is established and the patch is in whole cell mode, manipulating the IC environment by pipette solution exchange is a technical feat few can achieve (reference).  

This is where the intracellular exchange (ICE) assay developed by Evotec on Sophion Qube changes the game. 

The assay design: 

As is illustrated below (fig. 1), an EC salt solution is added to each well in a 384-well QChip via an inlet well and the same can be removed via a waste well, both from the top (fig. 1). On the other hand, the IC salt solution has only one inlet/outlet channel i.e. for both perfusing in and out. 

In the ICE assay, cells are patched on this QChip in the whole-cell mode using a negative pressure via the patch hole. The cells are thus surrounded by the EC solution while the cytosol is accessed via the IC solution.  

Figure 1-1

Figure 1: Architecture of each well in a 384-well Qchip (left). A single hole Qchip with the indicated intracellular (IC) solution inlet.


Hereafter, the magic begins: after one or more stabilization stages to obtain baseline activity, the IC solution is completely replaced with a new IC solution in sequential liquid periods. Our ICE assay has been developed to execute such IC exchanges up to four times (fig. 2), replacing the previous solution completely with a new solution each time.  

Figure 2

Figure 2: Liquid periods in the ICE assay with the stabilization periods to obtain baseline activity followed by four rounds of IC solution exchange. 


Thus, modulators can be delivered to the cytosolic face of the ion channel cumulatively.  

All the while, the cells are maintained in whole cell configuration in the QChip and the seal resistance does not drop below the QC threshold (40 MΩ, fig. 3). The ion channel target is observed to respond to each increasing concentration of the modulator post-exchange (fig. 3). 

Figure 3

Figure 3: (a) Seal resistance during sequential liquid periods interspersed with removal of the Qchip from the bio-chip interface, (b) Current vs time (IT) plot. Signal from the target ion channel responds to cumulatively increasing concentration of the modulator in cytosolic side. 


Achieve more with less reagents and resources: 

  • With four rounds of exchange, the volume of the IC solution required is only 4 ml for 3-4 experiments.  
  • The assay has a throughput of up to 26 compounds per Qchip for a 3-point dose response profiling.  
  • The assay is very resource-friendly also in terms of chemistry, since only minute amounts of compound are required. 
  • An experiment with four exchanges lasts only about 90 minutes.  

In a proprietary assay developed by Evotec, it was possible to profile about 700 compounds in 70 runs performed over 64 days.  

Key learnings: 

  • The IC exchange assay can cumulatively test three increasing concentrations of the same compound, delivered to the cytosolic face, providing pre- and post-compound data.  
  • A complete solution exchange is possible between each concentration, thus preventing compound cross-contamination.  
  • The assay can also be adapted to different intracellular ion concentrations, pH values, or signaling molecules.  
  • Druggability of ion channels as a family of proteins is greatly enhanced. 

No matter whether your drug discovery program is in the hit ID, hit-to-lead, lead optimization or early safety assessment, the ICE assay is useful for high-throughput hit profiling at any point.  

Companies interested in using Evotec’s technology are welcome to contact Evotec at