Great talking to you today, Markus. As we are going to talk about the patch clamp chips, let's maybe start with you telling us a bit about what those chips actually are and why they are important.

Sure. So, the planar patch clamp chips (also called biochips, microchips, or electrodes) are essentially small devices that allow for ion transport measurements without the use of classical patch pipettes. To my knowledge, those chips are the core elements of every automated patch clamp (APC) solution existing today.

As it goes from their name, planar chips comprise a planar substrate with one or multiple micron-sized holes in it. Such a design allows replacing the classically used glass pipette, greatly reducing the need for manual dexterity and facilitating patch clamp recordings… plus offering a number of advantages in terms of the experimental parameters (lower capacitance, better access resistance).

You know, the invention and further development of planar patch clamp chips was an absolutely crucial step for the advancement of automated patch clamp approaches. I don't think I can overstate it. The patch pipettes, the traditional glass electrodes, with all their advantages, were at the same time sort of a bottleneck for automated high throughput patch clamp. All the early pipette-based automated patch clamp systems never managed to achieve commercial success and were withdrawn from the market.

When talking about the invention of planar patch clamp chips, do you mean the late 1990s, when several companies were developing the first planar patch clamp systems?

Well, yes and no. So, the idea of the planar patch clamp has been known since the 1970s when Ukrainian scientists, led by Platon Kostyuk, used this approach for the first time to record transmembrane currents in big molluscan neurons. Basically, if you look at their Nature paper, they presented maybe the first planar patch clamp chip with some microfluidics capabilities, allowing for intracellular dialysis during recordings ¹. Well, their device was only suitable for huge cells, the substrate was made from polyethylene film, and the hole was approximately 40 micrometers in size, but still, it was working. That's why I consider this device a kind of "forefather" of the modern planar patch clamp chips.

But you know what, I think that the timing was not the best for that discovery. They published their paper in 1975, but already in 1976, Erwin Neher and Bert Sakmann developed the pipette-based patch clamp technique and published their first recordings of acetylcholine receptor single-channel currents ². This paper kind of “overshadowed” the earlier discovery of Kostyuk et al. Also, at that time there was no need or demand for recording currents from multiple cells simultaneously, neither were ion channels unraveled as individual drug targets. So, their discovery did not receive the attention it deserved... until the late '90s when the need and the demand emerged.

As you can see, the basic concept has been there for a while. Having said that, I think that all the work that has been done in the late '90s - early 2000s to make planar chips suitable for mammalian cells was equally crucial for the development of automated patch clamp to the level it is at now.

Could you elaborate on this, please? What developments have been done to make planar chips suitable for mammalian cells?

Firstly, different labs and companies jumped on the search for the best substrate. Kostyuk et al. were using polyethylene film as the substrate. It seems that the adhesion of cells to this substrate wasn't perfect, so to reduce the leak, they covered the walls of the pore with an adhesive material made from Parafilm. As far as I know, this substrate has not been further adopted by others.

In the early 2000s, we started to see reports on substrates made of fused quartz, borosilicate glass, silicone, silicone polymer PDMS, plastics... They all have their advantages and disadvantages, but the ideal substrate for the planar patch clamp chip should possess certain characteristics: it should have excellent dielectric properties; the cells should be able to adhere to it; and finally, the chip should have low capacitance to enable high-quality low-noise recordings.

Along with the substrate, another important characteristic of the planar chip is chip resistance, which is defined by the diameter of the hole we make in a substrate. And I should tell you that it could easily become a challenge if you need to make micron-sized holes in any substrate, especially if you need smooth round edges, free from any contamination. And that is exactly what we need to get the good seal.

Different approaches are used to "drill" the hole in different substrates. For example, silicone-based substrates can be rather readily micro-molded, so you can have dedicated molds for different hole sizes. In contrast, making a hole in glass requires more sophisticated micro-structuring approaches, such as ion-track etching.

I told you about the substrate and the hole, and the last thing I wanted to mention here is microfluidics. There have also been some developments in this area, and now, along with open-well chips, you can find planar chips with built-in microfluidic channels.

To conclude, I want to emphasize that a lot of work has been done in this area since the late 90s. Now, we have a better understanding of the electrical and adhesive properties of different substrates, we know how to make apertures of different sizes in these substrates, and we know how to better control solution exchange. But I must tell you that chip development still continues nowadays as we see new projects with new requirements. And, so, it's still an active area with a lot of R&D.

What about Nanion? Do you do chip R&D at Nanion?

Oh yes, very much. All our chips we develop and produce ourselves here in Munich, at Nanion headquarters. This was the case in 2002, when Niels and Michael started the company, and it’s still the case now. The only difference is that in 2002 Nanion produced chips for the Port-a-Patch only, and now we produce a wide range of chips for different instruments and different applications.

We do have people here who devote 100% of their time to chip development and/or production. Our Chip Department now comprises two teams: Chip Development Team and Chip Production Team, totaling together 15 people, mostly engineers. So, I would say it’s quite big. But there’s a reason why we have so many people involved in our chip development and production: our customer base is growing, so more and more chips are to be delivered every year for each of our instruments. And we need to ensure that the chips delivered are of the right quality, the right amount and arrive right on time. On top of that, we have quite a few chip development projects initiated following specific requests from our customers.

So, yes, chip development and production is something that holds a very important place at Nanion.

Could you walk us through the process of chip production at Nanion? If we were to create the chip for planar patch clamp ourselves, what would that look like?

My pleasure. First of all, you need the raw material, the substrate. I told you before that there are different substrates to choose from, and here at Nanion, we believe that the ideal substrate for planar patch clamp chips is borosilicate glass. We are using it for all our automated patch clamp devices as it represents the exact same glass that you use in manual patch clamp. This glass possesses all the characteristics I was telling you about: it has excellent dielectric properties; it’s possible to seal the cells with it, and our glass chips have very low capacitance (≤10 microF). On top of that, borosilicate glass is widely used and has been tested by all the patch clampers in the world. What better substrate could you think of?

From the start, Nanion developed extensive expertise in producing micro-structured glass chips. From the Port-a-Patch to the SyncroPatch 384, in all our instruments, we use glass chips.

Now, let’s get back to our chip manufacturing process. After choosing the substrate, you need to make it planar (in the end, you are going to produce planar chips, right?). For this, we have qualified partners who supply us with pure glass sheets and glass coverslips that we are using to produce the different consumable types for each of our products.

Once we’ve received glass sheets and coverslips, we assemble them with plastic parts that serve as solution compartments. This process includes various quality control (QC) steps and raw materials are mounted within a robotic assembly line (similar to what you may imagine from big industrial processing setups), leading to raw plates which are then processed further…

For every instrument, this plastic part could be different, but for you to get an idea, it’s essentially a bottom-less 384-well plate. What we do next is we fix the glass sheet in place of the missing bottom. The result is the 384-well plate with a glass bottom.

Additional QC steps follow at this stage, every plate gets a unique barcode identifier, and all the quality control information is stored for every chip until the customer makes a recording. This makes our process completely transparent, and we can trace back every step.

If the plate passes the QC, the raw chip is ready for the perforation processing. Here, we can adjust our production lines to microstructure one or multiple holes in each well of the raw plate, depending on the specifications (such as patch hole size). The processing follows strict control limits and is re-calibrated for every product. As I told you before, all information is stored and associated with the individual barcode, so that one could actually go back to each individual well/patch clamp hole to see its performance. For every chip type, we can adjust the parameters that we control. I’m talking not only about obvious parameters such as patch hole size, but also about more technical processing parameters that allow us to have full understanding and control of the process.

After this step, our plate becomes our beloved planar patch clamp chip, and after passing the final QCs, the chip is vacuum packed in a sealed container under climate-controlled conditions and is ready to be sent to the customer.

The whole process is essentially the same for every patch clamp chip type that we make.

Now you know everything.

So, you're saying that with micro-structuring, you can make one or multiple holes in each well. Are we talking about a fixed hole size, or can we change the hole size according to our needs?

Our vast experience and complete control over all production steps, brought us knowledge and competence to be super flexible, meaning we can customize our consumables to suit the exact customer needs and also shape them around new developments if needed. I would say this flexibility applies to 99.9% of the applications we work with. So, no matter what your specific needs are (in relation to the chips), we can tailor our devices to match them perfectly.

Regarding the hole sizes, we can make chips with small holes and chips with large holes. Just like in manual patch clamp (where you adjust the size of glass pipettes for different situations), you can use a bigger hole for better access or a smaller hole if you're worried about sealing. This flexibility is usually needed to accommodate variations in cell material.

A major recent development was the release of the 2^nd generation of consumables, where we combined very good sealing properties with improved access properties. We implemented a new raw chip type with reduced substrate thickness. Our thin glass version has been in the field for around two years now, and we have received very good feedback. I cannot thank the team enough for making this significant achievement possible in parallel with running production of the 1^st generation chips.

You know, when I tell you that we are super flexible, I mean it. It’s not only about the number of holes and the hole size. For example, very recently we have aimed to optimize the alignment of the patch clamp aperture and the pipetting tip. This could be relevant when working with low cell densities, making it more cost-efficient for the customers dealing with expensive cells.

Another recent development was the breakthrough that enables high throughput fluoride-free recordings in physiological solutions (without seal enhancers). This advancement brings our high throughput approach even closer to manual patch clamp. We presented this approach in detail in our publication on using physiological fluoride-free solutions for high throughput automated patch clamp experiments ³.

Oh, that’s interesting. Could you tell us more about seal enhancers and the fluoride-free approach?

Of course. So, you know that a good seal between the cell membrane and the substrate of the patch clamp chip is crucial for high-quality recordings. In APC this interaction is facilitated by the precipitation of insoluble salts, such as CaF₂. It is believed that this precipitation takes place at the interface between the cell and the pipette (or chip), thereby enhancing the sealing process. Therefore, these steps are commonly described as “seal enhancing”, where transiently elevated external calcium and intracellular fluoride form the precipitate. The approach is widely used in the APC world, but it is also frequently employed in manual patch clamp, when high sealing is needed. Besides CaF₂, it is also possible to work with other alternative insoluble salts, such as BaSO₄ (which means without fluoride), but still we work with precipitate.

I don't know who was the first to develop this approach, but I saw it for the first time in that same 1975 Nature paper by Kostuyk et al, where the first "planar patch clamp approach” was presented. In fact, that paper explained the effect of internal fluoride and phosphate on membrane currents. It showed that with fluoride or phosphate inside the cell, recordings and the leak conductance were stable for hours ¹.

This means that fluoride works very well, and it's widely used by electrophysiologists all over the world, especially by those studying voltage-gated sodium channels. However, I need to mention that fluoride has some known effects on the voltage-dependence of activation and inactivation of some Na_v channels. It also binds intracellular calcium and may interfere with G-protein coupled receptor or enzyme cascades. Therefore, while the use of fluoride in the internal solution is no problem for many experiments, there are situations where it is highly desirable to use fluoride-free internal solutions.

For these applications, we developed special NPC384-FF chips to allow fluoride-free experiments on the SyncroPatch 384. Although success rates are better when fluoride is used (which is expected), it’s clear that we have overcome the challenge of low success rates and low seal resistances on high throughput automated patch clamp devices when using fluoride-free physiological internal solutions. Furthermore, the approach does not depend on transiently elevated extracellular Ca²⁺ and can work constantly at physiological concentrations. I think it’s a great win, and we will continue to improve our FF chips to increase success rates even more.

At the beginning of our talk, you mentioned numerous developments related to planar patch clamp chips. One of these developments was the possibility of integrating microfluidic channels into the chips. However, for the SyncroPatch 384 chips, you decided to go with the open-well format. What are the advantages of this format over chips with integrated microfluidics?

As I mentioned before, since we produce all our chips in-house at Nanion, we have complete control over the production process and are very flexible. So, we manufacture both open-well chips (for the Port-a-Patch and the SyncroPatch 384) and chips with integrated microfluidic capabilities (for the Patchliner). Each of these formats has its own advantages and disadvantages.

The main advantage of microfluidics chips is the inbuilt control over solution exchange. However, after conducting multiple tests together with our customers, we decided to use the open-well format and have the microfluidics part reside in the recording chamber of the SyncroPatch 384. Why? Because this has several strong advantages that we and our customers find important.

Firstly, you can access it optically. This is a big advantage. You can take the chip out, and inspect the cells for fluorescence, for instance. Or you can combine your electrophysiological recordings with optical stimulation. Our engineers developed an Optogenetic Stimulation Tool as an add-on for the SyncroPatch 384, where cells can be stimulated by light of different wavelengths from the top of the recording chamber, allowing experiments with light-gated channels, such as channelrhodopsins (see, for example, our recent webinar on using the SyncroPatch 384 to characterize light-gated ion channels). As I mentioned earlier, recently we have optimized the alignment of the patch clamp aperture and the pipetting tip. This feature makes APC more cost-effective, as it facilitates working with low cell densities, and therefore is highly relevant when working with expensive cells. This is not possible with microfluidic chips.

Also, very recently, using a similar approach, we were able to develop an ultrafast pipetting flow method that can simulate shear stress/ mechanical activation of cells to elicit currents from mechanosensitive channels, such as PIEZO1. This development was the result of one of our close collaborations with academic partners and the work was just accepted in the Journal of General Physiology ⁴. It represents the first high-throughput mechanical stimulation with direct current read-out, and we will soon release this approach to our customers to hopefully accelerate research of mechanosensitive channels without the aid of chemical activators.

The open-well format is also advantageous when you want to record from big, non-symmetrical, and heavy cells, such as native cardiomyocytes. The design and geometry of modern microfluidics-based chips appear to prevent larger non-symmetrical cells from reaching and attaching to the patch clamp aperture. This has been confirmed in our recently published paper, in collaboration with the group of Professor Niels Voigt from the University of Göttingen. In the paper, we demonstrated for the first time the successful application of a high throughput APC system for the recording of action potentials and ion currents in freshly isolated mammalian atrial and ventricular cardiomyocytes. The open-well format was crucial here ⁵.

So you see, the open-well format presents quite a few important advantages over microfluidic-based chips. With careful planning and assay design, you also have very good control over solution exchange in open wells. But if you think that for your particular application you still need the solution control capabilities of a microfluidic system, we've got you covered with our Patchliner.

Thank you, Markus. That was a very interesting discussion, and our last question will be about the future. What future developments or advancements do you envision for planar patch clamp chips?

Hmm… in my opinion, modern patch clamp chips are already performing remarkably well in many applications. Having said that, it’s clear that for some applications, further developments are needed, particularly when it comes to fluoride-free recordings and primary cells. We already have quite high success rates with both fluoride-free recordings and primary cells, but we are working to make them even better.

Other developments that I think we’ll see in the future concern high throughput organellar patch clamp. Our chips have already been used to make recordings from lysosomes and mitoplasts on a Port-a-Patch. However, considering the increasing interest in this field, I believe that in the future, we’ll also have some high throughput organellar chips. So, watch this space.

References

1. Kostyuk PG, Krishtal OA, Pidoplichko VI. Effect of internal fluoride and phosphate on membrane currents during intracellular dialysis of nerve cells. Nature. 1975 Oct 23;257(5528):691-3. doi: 10.1038/257691a0
2. Neher E, Sakmann B. Single-channel currents recorded from membrane of denervated frog muscle fibres. Nature. 1976 Apr 29;260(5554):799-802. Doi: 10.1038/260799a0.
3. Rapedius M, Obergrussberger A, Humphries ESA, Scholz S, Rinke-Weiss I, Goetze TA, Brinkwirth N, Rotordam MG, Strassmaier T, Randolph A, Friis S, Liutkute A, Seibertz F, Voigt N, Fertig N. There is no F in APC: Using physiological fluoride-free solutions for high throughput automated patch clamp experiments. Front Mol Neurosci. 2022 Aug 22;15:982316. doi: 10.3389/fnmol.2022.982316. eCollection 2022.
4. Murciano N, Rotordam MG, Becker N, Ludlow MJ, Parsonage G, Darras A, Kaestner L, Beech DJ, George M, Fertig N, Rapedius M, Brüggemann A. A high-throughput electrophysiology assay to study the response of PIEZO1 to mechanical stimulation. J Gen Physiol. 2023 Dec 4;155(12):e202213132. doi: 10.1085/jgp.202213132. Epub 2023 Oct 6.
5. Seibertz F, Rapedius M, Fakuade FE, Tomsits P, Liutkute A, Cyganek L, Becker N, Majumder R, Clauß S, Fertig N, Voigt N. A modern automated patch-clamp approach for high throughput electrophysiology recordings in native cardiomyocytes. Commun Biol. 2022 Sep 15;5(1):969. doi: 10.1038/s42003-022-03871-2.