Vertical nanowire probes for intracellular signaling of living cells
- Ki-Young Lee†1, 2,
- Ilsoo Kim†1,
- So-Eun Kim1,
- Du-Won Jeong3,
- Ju-Jin Kim3,
- Hyewhon Rhim4,
- Jae-Pyeong Ahn5,
- Seung-Han Park6 and
- Heon-Jin Choi1Email author
© Lee et al.; licensee Springer. 2014
Received: 16 October 2013
Accepted: 22 January 2014
Published: 3 February 2014
The single living cell action potential was measured in an intracellular mode by using a vertical nanoelectrode. For intracellular interfacing, Si nanowires were vertically grown in a controlled manner, and optimum conditions, such as diameter, length, and nanowire density, were determined by culturing cells on the nanowires. Vertical nanowire probes were then fabricated with a complimentary metal-oxide-semiconductor (CMOS) process including sequential deposition of the passivation and electrode layers on the nanowires, and a subsequent partial etching process. The fabricated nanowire probes had an approximately 60-nm diameter and were intracellular. These probes interfaced with a GH3 cell and measured the spontaneous action potential. It successfully measured the action potential, which rapidly reached a steady state with average peak amplitude of approximately 10 mV, duration of approximately 140 ms, and period of 0.9 Hz.
KeywordsSilicon nanowire Intracellular interfacing Living cell Nanowire probe Action potential
The probing of an electrical activity in extracellular and intracellular modes at a single-cell level is crucial for understanding the whole nervous system [1–5]. In this respect, neuro-physiologists have investigated a small number of cells that are grown in defined patterns, allowing for the stimulation and recording of electrical activity of individual neurons [6–9]. However, these approaches are limited in precisely probe neural activity on a single-cell level. Conventional methods of electrophysiological measurement, which use micro-size electrodes such as electrolyte-filled glass pipettes and metal wires, are useful for identifying the electrical activity of electrogenic cells with a good signal-to-noise ratio and temporal resolution [10–12]. For all these advantages, it is difficult to achieve long-term signaling, repetitive monitoring, and multi-site recording. Other alternatives, such as multi-electrode arrays and planar FET devices [13–16], also have limitations in terms of the size of the probes used for signaling cell activity without cell damage.
Meanwhile, nanomaterials can potentially be exploited to achieve ultra-high sensitivity for various label-free biosensing applications as well as in direct probing of living cell activities [17–20]. Among nanomaterials developed to date, nanowires in particular have high aspect ratios, surface areas, and very small diameters on a sub-100-nm scale. Thus, they are ideal building blocks for probing single cell activity on a submicron scale. Notably, few studies have probed electrical activity (i.e., action potential) in an extracellular mode by using horizontal nanowire transistors [7, 21]. Probing the neural activity in an intracellular mode is also promising because the nanowire size is sufficiently small to provide an intracellular interface with neural cells without cell damage [22, 23]. Herein, we report the interfacing of neural cells with vertical Si nanowires and the probing of neural activity in an intracellular mode on a single-cell level.
Synthesis of nanowires
Vertical Si nanowires were grown on Si substrates using a vapor–liquid-solid mechanism with the assistance of Au colloid particles using a low pressure chemical vapor deposition process employing SiH4 as a silicon source [24, 25]. Based on the findings of previous studies [26, 27], the length (3 to 4 μm) and the diameter (60 to 100 nm) of the nanowires were set to optimum cell interfacing conditions.
Cell culture and fixation
An autoclave and ethanol were used to sterilize the substrates, and the substrate surfaces were chemically modified by a poly-L-lysine (PLL) coating for cell adhesion. Primary hippocampal neurons, or GH3 cells, were cultured on the substrates in a 24-well plate at 37°C in a 5% CO2 incubator. They were observed using a scanning electron microscope (SEM) and treated via a critical point drying technique after glutaraldehyde (for fixation) and osmium tetroxide (for contrast enhancement) treatments.
Results and discussion
Si nanowires were chosen as building blocks to probe neural cells because crucial factors for intracellular interfacing, such as their diameter, length, etc., can be easily tuned. Moreover, our previous study indicated that Si nanowires are bio-compatible to excitable cells (hippocampal neurons) and are thus safe for interfacing .
To verify how nanowire interfacing affects the cell viability, an MTT assay, a technique widely used to measure cell viability, was performed under the same conditions. Additional file 1: Figure S2 shows that the activity of the GH3 cell interfaced with a certain nanowire density and culture time is higher than that cultured on the bare silicon substrate. It also shows that too many interfaces with nanowires can have an adverse effect on the cell viability.
We investigated the effect of the population density of the nanowires on the growth of primary hippocampal neurons. At low nanowire density, as shown in Additional file 1: Figure S3d of supplementary data, hippocampal neurons displayed a normal morphology equivalent in quality to grown on the flat substrate. Their processes are well-developed in number and size. The figures also show that the nanowires penetrated the neural body. Under this intracellular interfacing, the entire cell membrane is complete and undamaged, retaining a structural functionality despite the distinct penetration of nanowires from the bottom to the top of the neuron cells. In the case of moderate density, hippocampal neurons failed to withstand wiring damage, as shown in Additional file 1: Figure S3e of supplementary data. The figure shows that many cells were destroyed, losing their original shape. The cell debris was tangled with nanowires in many locations. This indicates that the primary cell had grown and developed for some time after cell seeding. On the substrate with the highest nanowire density, hippocampal neurons showed no growth and remained embryonic in shape (Additional file 1: Figure S3f of supplementary data). This reveals that cells have specific tolerance toward the amount of nanowire penetration. GH3 cells are more active and thus are not as sensitive to the density of the nanowires as hippocampal neuron cells.
Previous studies indicate that probing cells using electronic devices are highly sensitive to the types of interfaces, as the most critical point in signal transfer from the cell to the device is the interface between these two domains [31–34]. In particular, the interface should have no cleft in order to allow signal transfer. The intracellular interfaces between nanowires and cells have not been investigated, and thus, these were examined in this study. Additional file 1: Figure S4a of supplementary data shows a schematic drawing of the cross-sectioning process. The intracellular coupled interfaces were cross-sectioned parallel to the longitudinal direction of the nanowires using a high-resolution Cross Beam focused ion beam field emitted SEM (FIB-FESEM). The sidewall was polished with a low ion current and imaged by SEM in an in situ mode. Additional file 1: Figure S4b of supplementary data shows a SEM image of the neuron-nanowire interface from the cross-section parallel to the longitudinal direction of the nanowires. The entire cross-sectional interfacial structure was well preserved, and distinct shrunken artifacts were not found. The nanowire penetrated the neuron membrane, which is attached tightly to the nanowires. These outcomes indicate that Si nanowires with diameters of <100 nm, lengths of several micrometers, and approximate densities of 2.5 × 104 mm−2 can achieve intracellular interfacing with excitable cells in a living state with tight interfaces without any cleft. This result implies that they may be suitable for probing excitable cells in an intracellular mode. Meanwhile, CNT array properties, i.e., conductivity, diameter, and length, are difficult to control for them to suit the intracellular interfacing or single cell signaling experiments. Patch clamp is conventional equipment for intracellular single cell signaling. The probe size of patch clamp is micro-scale, and the cell membrane should be broken for the probe and cell interfacing. Therefore, patch clamp is not suitable for in vivo experiment and neuronal interfaces between neuron.
Cr/Pt electrodes, which are connected with an external circuit, were then defined using photolithography and a sputtering process. A Pt layer that acts as an active electrode for signaling was subsequently defined for the individual nanowires by e-beam lithography and a sputtering process (shown in Figure 2c). This step was necessary because Si nanowires have a native SiO2 layer with thickness of 2 nm. This layer would build a very high potential barrier for signal transfer between the cell and nanowire probe. A second SiO2 layer was deposited via an HDP CVD process in order to electrically isolate the entire electrode from buffer solution and protect the electrodes from the chemical reaction in the wet cell culturing medium. Finally, the second passivation layer on the top part of nanowire probe was etched selectively by blocking the rest of the probe, which was wrapped with polymethyl methacrylate. This anisotropic wet etching method makes the nanowire probe have a suitable structure for intracellular recording (shown in Figure 2d).
Here, n is the number of electrons transferred during the electrochemical process, F is Faraday's constant, D and C are the diffusion coefficient and concentration of the electroactive species respectively, l and r are the length and radii of nanoelectrode, respectively, and t is time scale of the CV experiment, which is represented by RT/Fv. The experimental limiting current value at our nanoelectrode is 4.5 nA, which is similar to the theoretical limiting current value (4.21 nA/μm).
After signal recording, the coupled vertical nanowire probe-cell was investigated to clarify whether the nanowire probe penetrates the GH3 cell, which is essential for intracellular signaling. Figure 3c shows the cross-section of the coupled interface prepared by a high-resolution Cross Beam FIB-FESEM. Two passivation layers that coated the nanowires and a Pt layer for signal collection at the tip of the nanowires can be clearly seen in the cross-section. It is noted that the nanowire probe pierced through the cellular membrane in a bent shape, possibly due to compression by the weight of the cells. A robust passivation layer also acts as a buttress, which supports a nanowire against the cell. Figure 3c also shows that the membranes of the cells perforated by the vertical nanowire probe adhere closely to the top passivation layer without any voids. This tight coupling of the membrane and the SiO2 layer prevent the cytoplasm of the GH3 cell from mixing with the culture medium and the standard bath solution. By thus isolating the cells physically, it is possible to record the electrical activity inside of the cell in an intercellular mode.
We demonstrated a vertical nanowire probe can be used as a tool for intracellular probing of the electrical activity of single cells. The results indicate that interfacing of vertical grown nanowires with neuronal cells (i.e., intercellular penetration), which is essential to probe living cells in an intracellular mode, can be successfully achieved by controlling the diameter, length, and density of the nanowires. It has been demonstrated that the device structure, which consisted of passivation layers and signal collector layers, is mechanically robust and can overcome the mechanical resistance from the cells and is also electrically workable for probing the action potential. It is also shown that intracellular signaling is possible, because the nanowire probe is interposed in the GH3 cell and the cell membrane is tightly attached to the passivation layer. There have been previous studies involving vertical nanowire array electronic devices [40–42] indicating the feasibility of producing vertical nanowire probes on a large scale. The outcomes of this study can be easily extended to the signaling of neural networks such as cultured primary neurons or brain slices, where it is necessary to measure long-term cellular activity in a large working area [43, 44].
This work was supported by the National Research Foundation of Korea (NRF) grant, funded by the Korea government (MEST) (no. 2012R1A2A1A03010558) and the Pioneer Research Program for Converging Technology (no. 2009-008-1529) through the Korea Science and Engineering Foundation funded by the Ministry of Education, Science & Technology.
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