Optimizing Nanoelectrode Arrays for Scalable Intracellular Electrophysiology

Abstract

Electrode technology for electrophysiology has a long history of innovation, with some decisive steps including the development of the voltage-clamp measurement technique by Hodgkin and Huxley in the 1940s and the invention of the patch clamp electrode by Neher and Sakmann in the 1970s. The high-precision intracellular recording enabled by the patch clamp electrode has since been a gold standard in studying the fundamental cellular processes underlying the electrical activities of neurons and other excitable cells. One logical next step would then be to parallelize these intracellular electrodes, since simultaneous intracellular recording from a large number of cells will benefit the study of complex neuronal networks and will increase the throughput of electrophysiological screening from basic neurobiology laboratories to the pharmaceutical industry. Patch clamp electrodes, however, are not built for parallelization; as for now, only ∼10 patch measurements in parallel are possible. It has long been envisioned that nanoscale electrodes may help meet this challenge. First, nanoscale electrodes were shown to enable intracellular access. Second, because their size scale is within the normal reach of the standard top-down fabrication, the nanoelectrodes can be scaled into a large array for parallelization. Third, such a nanoelectrode array can be monolithically integrated with complementary metal-oxide semiconductor (CMOS) electronics to facilitate the large array operation and the recording of the signals from a massive number of cells. These are some of the central ideas that have motivated the research activity into nanoelectrode electrophysiology, and these past years have seen fruitful developments. This Account aims to synthesize these findings so as to provide a useful reference. Summing up from the recent studies, we will first elucidate the morphology and associated electrical properties of the interface between a nanoelectrode and a cellular membrane, clarifying how the nanoelectrode attains intracellular access. This understanding will be translated into a circuit model for the nanobio interface, which we will then use to lay out the strategies for improving the interface. The intracellular interface of the nanoelectrode is currently inferior to that of the patch clamp electrode; reaching this benchmark will be an exciting challenge that involves optimization of electrode geometries, materials, chemical modifications, electroporation protocols, and recording/stimulation electronics, as we describe in the Account. Another important theme of this Account, beyond the optimization of the individual nanoelectrode-cell interface, is the scalability of the nanoscale electrodes. We will discuss this theme using a recent development from our groups as an example, where an array of ca. 1000 nanoelectrode pixels fabricated on a CMOS integrated circuit chip performs parallel intracellular recording from a few hundreds of cardiomyocytes, which marks a new milestone in electrophysiology.

High‐Aspect‐Ratio Nanostructured Surfaces as Biological Metamaterials

“…The use of electroporation to facilitate intracellular electrical access is common throughout the literature [87,94,[379][380][381] and is consistent with the use of electroporation to enhance intracellular delivery, as discussed above. Some have argued that this is problematic when studying neuronal networks, [95,380] as electroporation overly perturbs the electroanatomy of the cells under investigation [383] and can cause damage to the nuclear membrane. [343] Indeed, Hai and Spira have proposed that electroporation on nanostructures can itself be a technique to study membrane repair dynamics.…”

“…Very large scale integration (the name given to the process of integrating millions of discrete electrical components into a single silicon chip) can also be used to help rapidly upscale the number of electrodes . Park and co‐workers have illustrated this approach through the development of an array of 1024 electrodes, each topped with nine titanium/platinum‐coated silicon dioxide nanowires that they used to interface with cells to sense and stimulate electrical activity . They used this platform to record network‐level (collective cell behavior) signals and synapse connections in rat cortical neurons and cardiac cells .…”

“…While single nanostructures can address single cells, and single nanostructures can be individually addressed, reconciling both of these behaviors remains a significant fabrication challenge. In particular, when studying network behavior, deconvoluting the complex signal behaviors in confluent cultures of electrogenic cells remains an open area of research …”

“…[379] Park and co-workers have illustrated this approach through the development of an array of 1024 electrodes, each topped with nine titanium/platinum-coated silicon dioxide nanowires that they used to interface with cells to sense and stimulate electrical act ivity. [28,94,370,380] They used this platform to record network-level (collective cell behavior) signals and synapse connections in rat cortical neurons [28] and cardiac cells. [94] Similarly, Braeken et al fabricated an array of 16 384 individually addressable tungsten/ silicon dioxide/tin nitride sub-cellular electrodes, albeit with relatively low aspect ratios (≈1.3:1).…”

“…In particular, when studying network behavior, deconvoluting the complex signal behaviors in confluent cultures of electrogenic cells remains an open area of research. [380]…”