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.
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.
- Hamill OP, Marty A, Neher E: Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflug Arch Eur J Phy 1981, 391: 85–100. 10.1007/BF00656997View ArticleGoogle Scholar
- Markram H, Lübke J, Frotscher M, Sakmann B: Regulation of synaptic efficacy by coincidence of postsynaptic APs and EPSPs. Science 1997, 275: 213–215. 10.1126/science.275.5297.213View ArticleGoogle Scholar
- Marom S, Shahaf G: Development, learning and memory in large random networks of cortical neurons: lessons beyond anatomy. Q Rev Biophys 2002, 35(1):63–87.View ArticleGoogle Scholar
- Stuart G, Spruston N, Sakmann B, Häusser M: Action potential initiation and backpropagation in neurons of the mammalian CNS. Trends Neurosci 1997, 20(3):125–131. 10.1016/S0166-2236(96)10075-8View ArticleGoogle Scholar
- Bean BP: The action potential in mammalian central neurons. Nat Rev Neurosci 2007, 8: 451–465. 10.1038/nrn2148View ArticleGoogle Scholar
- Fromherz P: Electrical interfacing of nerve cells and semiconductor chips. Chem Phys Chem 2002, 3(3):276–284. 10.1002/1439-7641(20020315)3:3<276::AID-CPHC276>3.0.CO;2-AGoogle Scholar
- Eschermann JF, Stockmann R, Hueske M, Vu XT, Ingebrandt S, Offenhäusser A: Action potentials of HL-1 cells recorded with silicon nanowire transistors. Appl Phys Lett 2009, 95: 083703. 10.1063/1.3194138View ArticleGoogle Scholar
- Gabay T, Jakobs E, Ben-Jacob E, Hanein Y: Engineered self-organization of neural networks using carbon nanotube clusters. Physica A 2005, 350: 611–621. 10.1016/j.physa.2004.11.007View ArticleGoogle Scholar
- Zheng B, Hsieh S, Wu CC, Wu CH, Lin PY, Hsieh CW, Li IT, Huang YS, Wang HM, Hsieh S: Hepatocarcinoma single cell migration on micropatterned PDMS substrates. Nano Biomed Eng 2011, 3: 99–106.Google Scholar
- Bi GQ, Poo MM: Synaptic modifications in cultured hippocampal neurons: dependence on spike timing, synaptic strength, and postsynaptic cell type. J Neurosci 1998, 18: 10464–10472.Google Scholar
- Yoshimura M, Nishi S: Blind patch-clamp recordings from substantia gelatinosa neurons in adult rat spinal cord slices: pharmacological properties of synaptic currents. Neuroscience 1993, 53: 519–526. 10.1016/0306-4522(93)90216-3View ArticleGoogle Scholar
- Akaike N, Harata N: Nystatin perforated patch recording and its applications to analyses of intracellular mechanisms. Jap J Physiol 1994, 44: 433–473. 10.2170/jjphysiol.44.433View ArticleGoogle Scholar
- Beggs JM, Plenz D: Neuronal avalanches in neocortical circuits. J Neurosci 2003, 23: 11167–11177.Google Scholar
- Maher MP, Pine J, Wright J, Tai YC: The neurochip: a new multielectrode device for stimulating and recording from cultured neurons. J Neurosci Meth 1999, 87: 45–56. 10.1016/S0165-0270(98)00156-3View ArticleGoogle Scholar
- Offenhäusser A, Sprössler C, Matsuzawa M, Knoll W: Field-effect transistor array for monitoring electrical activity from mammalian neurons in culture. Biosens Bioelectron 1997, 12(8):819–826. 10.1016/S0956-5663(97)00047-XView ArticleGoogle Scholar
- Liu H, Shen G: Ordered arrays of carbon nanotubes: from synthesis to applications. Nano Biomed Eng 2012, 4: 107–117.View ArticleGoogle Scholar
- Maxwell DJ, Taylor JR, Nie S: Self-assembled nanoparticle probes for recognition and detection of biomolecules. J Am Chem Soc 2002, 124: 9606–9612. 10.1021/ja025814pView ArticleGoogle Scholar
- Portney NG, Ozkan M: Nano-oncology: drug delivery, imaging, and sensing. Anal Bioanal Chem 2006, 384: 620–630. 10.1007/s00216-005-0247-7View ArticleGoogle Scholar
- McAlpine MC, Ahmad H, Wang D, Heath JR: Highly ordered nanowire arrays on plastic substrates for ultrasensitive flexible chemical sensors. Nat Mater 2007, 6: 379–384. 10.1038/nmat1891View ArticleGoogle Scholar
- Timko BP, Cohen-Karni T, Qing Q, Tian B, Lieber CM: Design and implementation of functional nanoelectronic interfaces with biomolecules, cells, and tissue using nanowire device arrays. IEEE Trans Nanotechnol 2010, 9: 269–280.View ArticleGoogle Scholar
- Patolsky F, Timko BP, Yu G, Fang Y, Greytak AB, Zheng G, Lieber CM: Detection, stimulation, and inhibition of neuronal signals with high-density nanowire transistor arrays. Science 2006, 313: 1100–1104. 10.1126/science.1128640View ArticleGoogle Scholar
- Tian B, Cohen-Karni T, Qing Q, Duan X, Xie P, Lieber CM: Three-dimensional, flexible nanoscale field-effect transistors as localized bioprobes. Science 2010, 329: 830–834. 10.1126/science.1192033View ArticleGoogle Scholar
- Schrlau MG, Dun NJ, Bau HH: Cell electrophysiology with carbon nanopipettes. ACS Nano 2009, 3: 563–568. 10.1021/nn800851dView ArticleGoogle Scholar
- Schmid H, Björk MT, Knoch J, Riel H, Riess W, Rice P, Topuria T: Patterned epitaxial vapor–liquid-solid growth of silicon nanowires on Si(111) using silane. J Appl Phys 2008, 2: 103.Google Scholar
- Kim I, Kim S-E, Han S, Kim H, Lee J, Jeong D-W, Kim J-J, Lim Y-B, Choi H-J: Large current difference in Au-coated vertical silicon nanowire electrode array with functionalization of peptides. Nanoscale Res Lett 2013, 8: 502–508. 10.1186/1556-276X-8-502View ArticleGoogle Scholar
- Lee KY, Shim S, Kim IS, Oh H, Kim S, Ahn JP, Park SH, Rhim H, Choi HJ: Coupling of semiconductor nanowires with neurons and their interfacial structure. Nanoscale Res Lett 2010, 5(2):410–415. 10.1007/s11671-009-9498-0View ArticleGoogle Scholar
- Kim W, Ng K, Kunitake ME, Conklin BR, Yang P: Interfacing silicon nanowires with mammalian cells. J Am Chem Soc 2007, 129: 7228. 10.1021/ja071456kView ArticleGoogle Scholar
- Bonifazi P, Fromherz P: Silicon chip for electronic communication between nerve cells by non-invasive interfacing and analog–digital processing. Adv Mater 2002, 14: 1190. 10.1002/1521-4095(20020903)14:17<1190::AID-ADMA1190>3.0.CO;2-#View ArticleGoogle Scholar
- Stett A, Müller B, Fromherz P: Two-way silicon-neuron interface by electrical induction. Phys Rev E 1997, 55(2):1779–1782. 10.1103/PhysRevE.55.1779View ArticleGoogle Scholar
- Merz M, Fromherz P: Silicon chip interfaced with a geometrically defined net of snail neurons. Adv Funct Mater 2005, 15(5):739. 10.1002/adfm.200400316View ArticleGoogle Scholar
- Schöning MJ, Poghossian A: Recent advances in biologically sensitive field-effect transistors (BioFETs). Analyst 2002, 127: 1137. 10.1039/b204444gView ArticleGoogle Scholar
- Poghossian A, Schöning MJ: Silicon based chemical and biological field effect sensors. In Encyclopedia of sensors. Vol 10. American Scientific Publishers; 2006:463.Google Scholar
- Schöning MJ, Poghossian A: Bio FEDs (Field-Effect Devices): state-of-the-art and new directions. Electroanalysis 1893, 2006: 18.Google Scholar
- Poghossian A, Schöning MJ: Chemical and biological field-effect sensors for liquids—a status report. In Handbook of biosensors and biochips. Willey-VCH; 2007:1. Ch 24 Ch 24Google Scholar
- Hunt JA, Williams DB: Electron energy-loss spectrum-imaging. Ultramicroscopy 1991, 38: 47. 10.1016/0304-3991(91)90108-IView ArticleGoogle Scholar
- Scherübl H, Hescheler J, Roy P: Steady-state currents through voltage-dependent, dihydropyridine-sensitive Ca2+ channels in GH3 pituitary cells. Soc B-Biol Sci 1991, 245: 127. 10.1098/rspb.1991.0098View ArticleGoogle Scholar
- Connollya P, Clarkb P, Curtisb ASG, Dowb JAT, Wilkinsona CDW: An extracellular microelectrode array for monitoring electrogenic cells in culture. Biosens Bioelectron 1990, 5: 223–234. 10.1016/0956-5663(90)80011-2View ArticleGoogle Scholar
- Aksay E, Gamkrelidze G, Seung HS, Baker R, Tank DW: In vivo intracellular recording and perturbation of persistent activity in a neural integrator. Nat Neurosci 2001, 4: 184–193. 10.1038/84023View ArticleGoogle Scholar
- Kipke DR, Vetter RJ, Williams JC, Hetke JF: Silicon-substrate intracortical microelectrode arrays for long-term recording of neuronal spike activity in cerebral cortex. IEEE Trans Neural Syst Rehabil Eng 2003, 11: 151–155. 10.1109/TNSRE.2003.814443View ArticleGoogle Scholar
- Rhim H, Baek HJ, Ho WK, Earm YE: The role of K+ channels on spontaneous action potential in rat clonal pituitary GH3 cell line. Kor J Physiol Pha 2000, 4: 81.Google Scholar
- Hochbaum AI, Fan R, He R, Yang P: Controlled growth of Si nanowire arrays for device integration. Nano Lett 2005, 5(3):457–460. 10.1021/nl047990xView ArticleGoogle Scholar
- Mohan P, Motohisa J, Fukui T: Controlled growth of highly uniform, axial/radial direction-defined, individually addressable InP nanowire arrays. Nanotechnology 2005, 16: 2903. 10.1088/0957-4484/16/12/029View ArticleGoogle Scholar
- Hsu CM, Connor ST, Tang MX, Cui Y: Wafer-scale silicon nanopillars and nanocones by Langmuir–Blodgett assembly and etching. Appl Phys Lett 2008, 93: 133109. 10.1063/1.2988893View ArticleGoogle Scholar
- Xie C, Hanson L, Xie W, Lin Z, Cui B, Cui Y: Noninvasive neuron pinning with nanopillar arrays. Nano Lett 2010, 10: 4020. 10.1021/nl101950xView ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.