By Tyler Shewbert
The first measurement of the action potential was performed using the voltage-clamp method in the 1950s on the squid axon. The patch-clamp method was developed in the 1970s, and using a gigaohm seal the ability to measure individual ion channels within mammalian neurons was attained [1, 2]. The limitations of the patch-clamp method are a lack of scalability that would allow researchers to perform simultaneous measurements of multiple neurons in vitro, both externally and internally, and its inability to perform longer data collection [3, 4]. Researchers have been studying ways to use nanoscale structures such as nanowires, nanopillars and other designs to help expand researchers’ ability to study individual neuron behaviors while also studying the behavior of surrounding neurons. This paper will report on two methods under development to either replace or augment the patch-clamp method and help further the understanding of neuroelectric behavior.
The patch-clamp method was developed by Bert Sakmann and Erwin Neher in the 1970s . The patch-clamp method involves using small, heat-polished pipettes with the electrodes of the size of 0.5-1.0 mm . To achieve the gigaohm seal with the cell membrane, which allows for accurate measurements of the action potential, extra care is taken to make sure the pipette is clean and suction is applied to the pipette interior . The resistance of the seal is inversely related to the signal to noise ratio, so the better the seal, the more accurate the ion channel recordings are . However, the patch-clamp method requires a skilled researcher to perform the method and is limited in its ability to study networks of cell electrical behavior in vitro .
Nanoscale structures have been explored as a way of performing these types of experiments. Vertical nanowire electrode arrays (VNEA), kinked nanowires or pillar-shaped nanowire with embedded pn-junctions, and other methods have been examined as possible methods .
A team at Harvard led by Hongkun Park developed a VNEA device with sixteen recording/stimulation pads. Each pad consisted of a 3×3 array of silicon nanowires (NW) that had dimensions of approximately 150 nm in diameter and 3 mm in length . The core of each wire was silicon and consisted of a metal tip to provide conductivity . Each array was a 4 mm square . This size was chosen because it is similar to the size of a neuronal cell so it was thought that would increase the chances of only one cell being connected to each array . The nanowires penetrated the cells membrane and recordings were performed . The seal was in the range of 100-500 MW . The following figure shows the a 3×3 pad:
Figure 1. A VNEA 3×3 pad. (from )
Charles Lieber’s research group has experimented with kinked nanowires and nanotubes (NTs) with FETs fabricated within the nanostructure. The NW or NT penetrates the cell membrane and the FET is used to record intracellular signals . The research discussed in this paper will discuss the use of SiO2 nanotubes to penetrate cells with embedded FETs for measuring the fast action potentials (FAPs) within the cell . Referred to as a branched intracellular nanotube FET (BIT-FET), the group was able to simulate FAPs in cells using tubes as small as 3 nm, much smaller than other methods . The nanotube connects the intracellular fluid to the FET as shown in the following figure:
Figure 2. Setup of the nanotube connecting the cytosol of the cell to the FET (from ).
Results and Discussion
The results of two recent papers will be discussed here. Both were published in 2012. The work of the team at Harvard led by Park using VNEA and Lieber’s team’s recent nanotube research. While the work of the Harvard team shows promise, the work of Lieber’s team with nanotubes has greater potential for solving the limitations of the patch-clamp method.
Park’s team performed a series of experiments on cultured cortical cells of rats . The pads of VNEA penetrated the cells of the rats . Patch-clamping was used to determine the membrane change, therefore determining if the VNEA had penetrated the membrane . In over half the instances the VNEA penetrated the cell allowing for recording and stimulation of the cell . Once the nanowire was inside of the cell, it was able to be stimulate and record the membrane potential using electrochemistry . The duration of stable recording was 10 minutes . A main advantage over external microelectrode devices is that the VNEA device was able to record multiple action potentials simultaneously .
Unfortunately, the VNEA devices had high impedance and the intracellular recording of the VNEA device provided no significant advantage over a method which uses mushroom-shaped, gold-tipped microelectrode devices externally . The high impedance issue could be solved by using more nanowires to penetrate the cell, in theory . Unfortunately, in practice other researchers have found that increasing the number of nanostructures for penetration on a pad has the effect of reducing the number of nanostructures that penetrate the cell, causing a “bed of nails” scenario .
The work using nano-FETs has proved more promising. This is because a recording that uses a FET built into the structure of the nanowire does not have to worry about impedance . The use of the BIT-FET recording intracellular signals was tested on embryonic chicken cardiomyocyte cells . After 45 seconds of the BIT-FET being in “gentle” contact with the cell membrane, the recorded electrical behavior showed a change that was consistent with the previously ran simulations that showed when intracellular recording took place . Full-amplitude action potential recording was performed and was reproduced . The BIT-FET devices had an hour of stable recording time .
They speculated that the penetration of the cell was spontaneous rather than forced since no external pressure has been applied when the recordings showed intracellular electrical behavior . They also found that the BIT-FET devices were reusable . The device was designed for intracellular, multiplex recording of cells, and this was confirmed . Due to their small size, the BIT-FET devices should be able to record electrical behavior from subcellular structures [3, 5]. These devices are limited at this point by the noise-levels of the nano-FET devices [3, 5]. The problem that other nano-FET devices have of having to push the cell onto the electrode seems to have found a solution in the BIT-FET device since no external pressure was being applied at the time of penetration [3, 5]. This was theorized to be caused by lipid fusion and has the benefit of a tight seal that removes the need for circuitry that dealt with probe-membrane leakage .
Outlook and relevance of work
The work performed by both teams contributed to the search for a method to replace or augment the patch-clamp method as a method of examining electrical behavior in cells. Between the two, the BIT-FET device and the method developed by Charles Lieber’s team was more promising. While VNEA devices successfully recorded intracellular signals from multiple cells, the nanostructure did not always penetrate the cell successfully. The penetration rate was actually reduced in similar style experiments when the number of structures had been increased to reduce the impedance in attempts to improve the signal to noise ratio .
The BIT-FET devices appear to be the route to a major breakthrough in intracellular recording. The ability of the BIT-FET to spontaneously penetrate the cell membrane helps to solve a problem that had been faced by kinked nanowires and other methods . The BIT-FET’s ability to record subcellular structures accurately has the potential to replace the patch-clamp method. Also, the ability for multiple action potentials across many cells to be recorded simultaneous, something that the VNEA devices were also able to do, is invaluable. If improvements in reducing nano-FET noise levels succeed, these devices might prove quite successful as a complement and eventual replacement to the patch-clamp method.
 Shewbert T. From the Voltage Clamp to the Patch-clamp. Santa Cruz: University of California, Santa Cruz; 2017. p. 5.
 Cui Y, Wei Q, Park H, Lieber CM. Nanowire nanosensors for highly sensitive and selective detection of biological and chemical species. Science. 2001;293:1289-92.
 Spira ME, Hai A. Multi-electrode array technologies for neuroscience and cardiology. Nature nanotechnology. 2013;8:83-94.
 Robinson JT, Jorgolli M, Shalek AK, Yoon M-H, Gertner RS, Park H. Vertical nanowire electrode arrays as a scalable platform for intracellular interfacing to neuronal circuits. Nature Nanotechnology. 2012;7:180-4.
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