| Notes |
Articles
https://doi.org/10.1038/s41565-019-0478-y
1
Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA. 2
Advanced Technology Institute, University of Surrey,
Guildford, Surrey, UK. 3
Center for Nanomedicine, Institute for Basic Science (IBS), Yonsei-IBS Institute, Yonsei University, Seoul, Republic of Korea. 4Center for Brain Science, Harvard University, Cambridge, MA, USA. 5
John A. Paulson School of Engineering and Applied Sciences, Harvard University,
Cambridge, MA, USA. 6These authors contributed equally: Yunlong Zhao, Siheng Sean You, Anqi Zhang *e-mail: cml@cmliris.harvard.edu
Developing new tools that enable reproducible high spatial–
temporal resolution recording of intracellular potential, while
maintaining the capability for device scalability, are key goals
for advancing electrophysiology studies of electrogenic cells and
cell networks1–4
. Patch-clamp electrodes have been the gold standard for cell electrophysiology for decades, and they have shown
that accurate recording of the intracellular potential requires a
high-resistance seal against the cell membrane and low resistance
between the recording element and the cell interior5,6
. Recent studies have focused on several solid-state nanodevice architectures,
including nanowire-based structures for optical neuronal stimulation7,8
, scalable on-chip micro/nano-structured electrode arrays9,10
for attenuated intracellular recording via electroporation11,12 and/
or optoporation13,14, and three-dimensional (3D) nanowire fieldeffect transistor probes for intracellular recording of single cells15,16.
Nanowire probes have recorded cardiac intracellular action potentials with amplitudes comparable to those recorded with patchclamp micropipettes15,16, but have relied on one-by-one fabrication
that has been difficult to scale up. For solid-state nanoprobes to
achieve comparable recording signal-to-noise ratio and amplitude to those of patch clamp measurements, the nanodevice must
achieve direct contact of the recording element with the intracellular solution without significantly disturbing the cell membrane2,9
.
Fulfilling these criteria requires understanding the size, geometry
and mechanical and biochemical factors present at the cell membrane–nanodevice interface. Recent work suggests that nanoscale
size and geometry play a key role in the interaction between the
nanostructure and the cell membrane17,18. For example, nanoscale
membrane curvature elevates the local concentration of endocytosis-related proteins17,18, influences the conformation and activity of
transmembrane proteins19 and is hypothesized to recruit a sequence
of proteins leading to membrane fission20. Building on these studies, we hypothesize that inducing appropriate nanoscale curvature
on the cell membrane via rational device design will facilitate probe
internalization and enable intracellular recording.
Here, we investigate how the size and geometry of nanoprobes influence intracellular recording by fabricating scalable 3D
U-shaped nanowire field-effect transistor (U-NWFET) arrays in
which both the radii of curvature (ROC) and active sensor sizes are
controlled (Fig. 1a). To investigate how these design factors affect
electrophysiological recording, arrays of U-NWFET probes fabricated from 15-nm-diameter p-type Si nanowires with ROC from
0.75 to 2μm and active channel lengths from 50 to 2,000nm were
used to probe cultured primary neurons and human cardiomyocytes. Schematically, we ask whether probes with the smallest ROC
and sensor size (Fig. 1b(i)) can facilitate recording full amplitude
intracellular action potentials and subthreshold features, where
increases in the ROC and detector sizes (Fig. 1b(ii)) would lead
to recording smaller amplitude intracellular-like or extracellular
action potential peaks.
U-shaped nanowire probe fabrication and characterization
Our strategy for producing reproducible arrays of U-NWFET
probes with controlled ROC and active FET channel lengths or
detector sizes involves two key techniques (see Methods for details).
First, large-scale, shape-controlled deterministic assembly21 is
used to produce U-shaped nanowire arrays from 15-nm-diameter
Si nanowires with controllable ROC on top of Si3N4 patterns
(Fig. 2a(i) and Supplementary Fig. 1a–e). Metal contacts are then
deposited and passivated by an upper Si3N4 layer (Supplementary
Fig. 1f). Second, we exploit spatially defined solid-state transformation22 to convert Si nanowire segments underneath and adjacent
to the Ni diffusion layer to metallic NiSi, thereby producing a controlled length of FET sensing elements at the tips of the U-shaped
nanowire probes (Fig. 2a(ii) and Supplementary Fig. 1g). Finally,
etching of the sacrificial layer allows the probes to bend upward
Scalable ultrasmall three-dimensional nanowire
transistor probes for intracellular recording
Yunlong Zhao 1,2,6, Siheng Sean You 1,6, Anqi Zhang1,6, Jae-Hyun Lee1,3, Jinlin Huang1
and
Charles M. Lieber 1,4,5*
New tools for intracellular electrophysiology that push the limits of spatiotemporal resolution while reducing invasiveness
could provide a deeper understanding of electrogenic cells and their networks in tissues, and push progress towards human–
machine interfaces. Although significant advances have been made in developing nanodevices for intracellular probes, current
approaches exhibit a trade-off between device scalability and recording amplitude. We address this challenge by combining
deterministic shape-controlled nanowire transfer with spatially defined semiconductor-to-metal transformation to realize scalable nanowire field-effect transistor probe arrays with controllable tip geometry and sensor size, which enable recording of up
to 100 mV intracellular action potentials from primary neurons. Systematic studies on neurons and cardiomyocytes show that
controlling device curvature and sensor size is critical for achieving high-amplitude intracellular recordings. In addition, this
device design allows for multiplexed recording from single cells and cell networks and could enable future investigations of
dynamics in the brain and other tissues.
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due to interfacial strain in the metal interconnects (Fig. 2a(iii))15,
yielding probe arrays with up to four addressable U-NWFETs per
bend-up probe arm (Supplementary Fig. 1h).
Optical microscopy and scanning electron microscopy (SEM)
were used to characterize key steps in the U-NWFET probe fabrication flow. Optical microscopy images of the patterned bottom
passivation layer and U-shaped trenches that set the ROC during
nanowire assembly (Supplementary Fig. 2a,b) as well as three probes
with U-shaped nanowires, metal contacts and top passivation layer
(Fig. 2b) are indicative of the deterministic parallel assembly of
U-shaped nanowire probes with defined ROC. Compositionsensitive SEM images of U-NWFETs following annealing of the
patterned Ni further highlight the control of channel lengths from
~50nm (Fig. 2c) to 500 and 2,000nm (Supplementary Fig. 2c,d).
Measured channel lengths and ROCs were found to be consistent
with those designed (Supplementary Fig. 3). Etching of the Ni
release layer produces arrays of probes, including single U-NWFET
probes (Fig. 2d) and multiple U-NWFET devices on a single probe
arm (Supplementary Fig. 2e) where the active U-NWFET sensor
elements are oriented upwards away from the substrate.
Electrical transport studies in air and aqueous solution were
carried out to characterize the sensor properties. Current versus
drain–source voltage (I–Vds) measurements on devices with channel lengths of ~50, 500 and 2,000nm (Fig. 2e–g; N=10, each channel length) in the dry state yield average conductances of 3.3±0.6,
0.7±0.2 and 0.3±0.1μS, respectively. In addition, conductance
versus water gate voltage (Vg) measurements in aqueous solution
(Fig. 2h–j) yield average transconductances of 5.4±1.3, 2.3±0.7
and 0.9±0.3μSV−1
for 50, 500 and 2,000nm channel lengths,
respectively. Plots of probe transconductance versus ROC
(0.75–2.0μm) for devices with 50, 500 or 2,000nm FET channels
(Supplementary Fig. 4 and Supplementary Table 1) show that transconductance does not significantly vary as a function of ROC in our
designed strain range (Supplementary Table 2). The conductance
and transconductance results for the U-NWFETs are roughly consistent with the expected inverse relationship to the channel length.
Finally, the transconductance and measured noise values yield an
estimate for signal detection sensitivity (three standard deviations)
of 0.90±0.60, 1.2±0.9 and 1.9±0.9mV for 50, 500 and 2,000nm
channel lengths, respectively, which should allow detection of the
typical 1–10mV subthreshold activities of neurons2
.
Near full amplitude intracellular recordings
With these characterization results, we first asked whether ultrasmall
U-NWFET probes could record full amplitude intracellular action
potentials from primary neurons. First, a single U-NWFET probe
with ~50nm FET length and 0.75μm ROC was used to sequentially measure six independent dorsal root ganglion (DRG) neurons (see Methods and Supplementary Fig. 5), where the probe was
not remodified with lipid between the sequential measurements
(Supplementary Fig. 6). In each trace, we observe a drop in the
baseline potential upon initial cell contact (Fig. 3a). Subsequently,
either sparse peaks (cells 1, 3 and 6) or periodic peaks (cells 2, 4 and
5) are observed with amplitude of 60–100mV and signal-to-noise
ratios of 115±29. For each cell, the recorded potentials have consistent shape and duration, and characteristic single peaks recorded
from the six cells are shown in Fig. 3b. An additional set of data
recorded from two DRG neurons without spontaneous firing properties showed a voltage drop (Supplementary Fig. 7a), or one single
peak followed by a voltage drop (Supplementary Fig. 7b), during
device penetration. Following the initial recording, we observed a
gradual decrease in the peak amplitude as well as a positive shift in
the baseline potential (Fig. 3c,d and Supplementary Fig. 7).
These recordings highlight several key features. First, the waveforms, amplitudes, firing patterns and signal-to-noise ratios of the
peaks are similar to our patch-clamp recordings of similarly cultured DRG neurons (Supplementary Fig. 8) and are consistent with
the reported heterogeneity of spontaneously firing DRG action
potential waveforms and spike patterns23. These data thus indicate that the ultrasmall U-NWFETs with biomimetic phospholipid
modification can provide high-resistance membrane seals, achieve
direct access to the cell interior, and yield faithful recording of the
intracellular potential. Notably, the data recorded from some DRG
neurons exhibit characteristics consistent with mechanosensitive
properties24, including an increase in action potential firing rate
(Fig. 3c,d) and firing of a single action potential (Supplementary
Fig. 7b) during formation of the device/cell junction. A limitation
of the recording is, however, the shift in baseline and decrease in
recorded action potential amplitude at later times (for example,
Fig. 3c). We suggest that these changes are due to either an elastic
response from the cytoskeleton, which gradually pushes the probe
out of the cell as suggested in other intracellular chemical delivery
experiments25, or mechanical instability of the measurement set-up.
a
Cytoplasm
FET
Cytoplasm
Extracellular
fluid
FET
Conducting
nickel silicide
Conducting
nickel silicide
b
i ii
Fig. 1 | Ultrasmall U-NWFET probe as a new approach for electrophysiology. a, Schematics of intracellular recording by a U-NWFET probe. The location,
size, geometry of each probe and the sensor size can be well modulated by a deterministic shape-controlled nanowire transfer technique and spatially
defined transformation of Si nanowire segments to NiSi, respectively. b, Schematics of two possible probe–cell interfaces. (i) Internalization and highresistance seal of a short-channel U-NWFET to the cell membrane enables high-amplitude recording. The sensitive p-type Si NWFET region and the
metallic NiSi region on the U-shaped nanowire are marked with red and blue-grey, respectively. The nanowire is shown modified with phospholipid.
(ii) Partial sealing/internalization of the U-NWFET with longer channel length/ROC results in attenuated intracellular-like action potential recording.
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Given the high signal-to-noise ratio for our measurements,
we asked whether it was possible to observe subthreshold activity. Notably, close examination of a representative trace from a cell
with irregular firing pattern (Fig. 3d) shows subthreshold features,
including a single ~5mV peak (Fig. 3e(i)) and a series of three small
peaks (Fig. 3e(iii)) immediately before the initiation of an action
–0.10 –0.05 0.00 0.05 0.10
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Fig. 2 | Fabrication and characterization of U-NWFET probes. a, Schematics of device fabrication. (i) Assembly of U-shaped nanowire devices on a
Ni sacrificial layer and bottom Si3N4 passivation layer; electrical contacts to the transferred U-shaped nanowires are made via deposition of Cr/Au/Cr
(1.5/60/60 nm) metal interconnects, where the relative Cr/Au/Cr thicknesses yield a built-in strain that bends the probe up upon release. (ii) Deposition
of the top Si3N4 passivation layer and the Ni diffusion layer followed by rapid thermal annealing to transform the Si nanowire segments underneath and
adjacent to the Ni diffusion layer to NiSi, thus generating a local FET at the tip of the U-shaped nanowire. (iii) Probes bending upward after etching the Ni
diffusion and sacrificial layers. b, Optical image of three devices following deposition of metal interconnects and before deposition of the nickel diffusion
layer. Inset, magnified view showing that a U-shaped nanowire is deterministically transferred to the device tip. Scale bar, 20 μm. c, SEM image of the
device after Ni diffusion. Scale bar, 500 nm. Inset, magnified SEM image of the dashed region showing the resulting local FET at the U-shaped nanowire tip.
Imaging with backscattered electrons (BSE) shows the Si (dark region) and NiSi (bright region) distribution on the U-shaped nanowire. Scale bar, 50 nm.
d, Optical image of the bend-up device array in water. Scale bar, 20 μm. e–g, Current versus drain–source voltage (Vds) traces for 10 devices in the dry state
for ~50 nm (e), ~500 nm (f) and ~2,000 nm (g) channel lengths. Insets, SEM images taken using the BSE mode of the local FET following removal of the
Ni layer. Scale bars, 50 nm (e), 0.5 μm (f), 0.5 μm (g). h–j, Left, conductance versus reference water gate potential (Vg) recorded from one representative
device for each channel length (~50 nm (h), ~500 nm (i) and ~2,000 nm (j)) in Tyrode’s solution. Right, box and whisker plots showing the distribution
(N= 10) of transconductance for devices of each channel length gated by a reference electrode in Tyrode’s solution. The blue triangle shows the mean of
the transconductances, the top and bottom edges of the box indicate the upper/lower quartiles and the whiskers indicate the highest and lowest measured
values, respectively.
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potential, as well as an ~3mV peak not associated with an action
potential spike (Fig. 3e(ii)); we recorded similar results with patchclamp (blue triangles, Supplementary Fig. 8). Previous multi-patchclamp studies have reported comparable subthreshold signals and
attributed them to excitatory postsynaptic potentials in which a presynaptic cell triggers the firing of the postsynaptic cell2,26. This suggests that our U-NWFET devices can measure biologically relevant
subthreshold signals and could be used for future studies of neural
connections and synaptic activity.
After achieving neuronal intracellular recording, we asked
whether the U-NWFET probes could be generalized to other
electrogenic cells. To answer this, we cultured human induced
pluripotent stem cell-derived cardiomyocytes (HiPSC-CMs) (see
Methods). Contact of a HiPSC-CM and a U-NWFET probe of
~50nm FET length and 0.75μm ROC (Supplementary Fig. 9a,b)
initially yielded a ~25mV drop in the baseline, followed by periodic
~50mV positive waveforms with a sharp rising phase (<50ms),
slow falling phase (~400ms) and frequency of 1.25±0.04Hz. The
second measurement of the same cell resulted again in a drop in
the baseline potential and initial ~50mV positive waveforms with
frequencies of 1.23±0.02Hz (Supplementary Fig. 9c,d). Notably,
the waveform frequency, amplitude and shapes during the first and
second entry remain similar and are consistent with reported cardiac action potentials27, suggesting that the U-NWFET probe is also
able to record the intracellular potential of cardiac cells, and that the
internalization process is minimally invasive.
Effect of nanowire geometry and sensor size on recording
We first investigated geometry effects by fabricating probe arrays
with ROCs ranging from 0.75 to 2.0μm (Fig. 4a and Supplementary
Fig. 10) with fixed ~50nm sensor sizes, carrying out ~30 measurements for each ROC from both DRG neurons and HiPSC-CMs.
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
0 0.2 0.4 0.6 0.8 1.0 1.2 1.4
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0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
6.06 6.08 6.10 14.30 14.35 14.40 19.56 19.58
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0 2 4 6 8 10 12 14 16 18 20
0 2 4 6 8 10 12 14 16 18 20
Fig. 3 | Intracellular recording of DRG neurons by the ultrasmall U-NWFET probe. a, Intracellular action potentials recorded sequentially from six
independent neurons using the same U-NWFET probe without remodification. The numbering of the cells indicates the order in which the measurements
occurred. The probe has a FET channel length of ~50 nm and ROC of 0.75 μm. b, Summary of recorded action potentials from the six different cells in
a in chronological order. The maximum signal amplitude remains similar between these measurements, showing that the probe is highly reusable. The
signal-to-noise ratio of these recordings is 115 ± 29 (N= 6). c, Intracellular recording trace from a cell with regular firing pattern showing a gradual increase
in baseline potential and decrease in action potential amplitude. d, Representative trace from a cell with irregular firing pattern; subthreshold activities
appear in regions outlined by blue dashed lines. e, Enlarged regions of three different areas from d showing different types of subthreshold activity,
highlighted by the red arrows and blue dashed regions.
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For DRG neurons, representative intracellular/intracellular-like
recordings were obtained by probes with 1μm and 1.5μm ROC
(Fig. 4b,c), showing maximum action potential amplitudes of
~35mV and ~12mV, respectively. The distribution of maximum
recording amplitudes from both cell types (Fig. 4d,e) shows the
average values for DRG/HiPSC-CM cells and number of successful
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8
0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8
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0.66 0.67 0.68 0.69 0.70 0.71 0.72
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10 ms 5 mV
DRG neuron DRG neuron
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20 mV
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Max. spike amplitude (mV)
c
h
i ii
Fig. 4 | Effect of size and geometry of U-NWFET probes on electrophysiological recordings. a, Optical image of U-NWFET probes with different ROC
before deposition of a Ni diffusion layer of 0.75 μm (i), 1 μm (ii), 1.5 μm (iii) and 2 μm (iv). Scale bars, 2 μm. b,c, Intracellular/intracellular-like recording
from a DRG neuron by a ~50 nm FET channel length probe with 1 μm (b) and 1.5 μm (c) ROC. Insets, magnified views of selected action potentials.
d,e, Plot of maximum recorded spike amplitude of recorded action potentials from DRG neurons (d) and HiPSC-CMs (e) versus ROC with fixed ~50 nm
FET length. Coloured bars indicate the maximum spike amplitudes measured in the given dataset. The statistical significances were obtained by comparing
the datasets below the ends of the black line using Student’s t-test. *P< 0.1, ****P< 0.0001. The blue letters ‘b’, ‘c’ and ‘i’ highlight the data points from
b, c and i. f, Scatter plot of maximum recorded spike amplitude of DRG neurons (i) and HiPSC-CMs (ii) for ~500 nm FET channel lengths with 0.75 μm
ROC. The statistical significances **** shown in i and ii were obtained by comparing the datasets in i and ii with datasets of ROC 0.75 μm in d and e,
respectively. g, Plot of maximum recorded spike amplitude of HiPSC-CMs with ~2,000 nm FET channel. Blue circles, intracellular data recorded from
U-NWFETs with 1.0 μm ROC. Yellow circles, intracellular data recorded from U-NWFET with 1.5 μm ROC. Red triangles, extracellular (EC) data recorded
from U-NWFETs with 1.5 μm ROC. Letter ‘h’ represents the data point for the trace shown in h. Note that for all scatter plots, ~30 measurements were
attempted for each U-NWFET channel length and ROC, and recordings that did not result in measurement of intracellular/intracellular-like or extracellular
action potentials were considered as 0 mV max spike amplitude and not shown for clarity but are summarized in Supplementary Table 3. h, Extracellular
recording from HiPSC-CMs by an ~2,000 nm FET channel length probe with 1.5 μm ROC. Inset, magnified view of the highlighted spike. i, Intracellular
recording from HiPSC-CMs by an ~50 nm FET channel length probe with 0.75 μm ROC.
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recordings (out of ~30 measurements) of 34±30 (N=24)/34±14
(N=31), 19±15 (N=25)/31±9 (N=6) and 16±7 (N=2)/21±9
(N=7)mV for 0.75, 1.0 and 1.5μm ROC, respectively. Interestingly,
the 2.0μm ROC probes did not yield successful recordings on
either cell type, indicating that increasing ROC correlates with
lower recorded maximum amplitudes. Measurement of device transconductance (Supplementary Fig. 11) and SEM images (Supplementary Fig. 12) indicate that device characteristics do not change
following measurement.
Second, we studied how sensor size affects recording by fabricating U-NWFETs with channel lengths of 500nm and 0.75 µm ROC
(Supplementary Fig. 10). Measurements made on both DRG neurons and HiPSC-CMs showed 8±8 (N=7) and 23±13mV (N=10)
maximum intracellular action potential amplitudes, respectively
(Fig. 4f). Furthermore, U-NWFETs with channel lengths of
~2,000nm and ROC of either 1.0µm or 1.5µm yielded maximum amplitudes of 21±12mV (N=5, blue circles) and 8.0mV
(N=1, yellow circle), respectively (Fig. 4g). Some of the 1.5µm
ROC, 2,000nm channel U-NWFETs recorded negative spikes with
maximum amplitudes of 4±1mV (N=4, red triangles), while no
successful recordings were achieved on DRG cells with 2,000nm
channel probes. A representative trace showing negative spikes
from a HiPSC-CM (Fig. 4h) highlights their periodic negative
short <3ms duration that contrasts with a representative intracellular recording27 obtained from a HiPSC-CM using a 0.75μm ROC,
50nm U-NWFET (Fig. 4i). The above data show that reducing the
ROC and FET channel length has a statistically significant correlation with increases in both the maximum measured action potential
amplitudes and number of successful recordings (Supplementary
Table 3). At the most extreme limit, devices with 2μm ROC no
longer record action potentials, although devices with 1.5µm ROC
and 2,000nm channel lengths recorded waveforms characteristic of
extracellular signals27,28. We hypothesize that the observed increase
in recording amplitude with decreasing ROC and channel length
is not solely from physical interactions between the device and the
cell but also a consequence of reported results demonstrating that
nanoscale curvature can induce activation of endocytosis and related
biological pathways17–20,29. Our studies indicate that using nanoscale
topography to enhance device uptake is critical for developing tools
that faithfully capture intracellular action potential features.
We also examined the relationship between U-NWFET device
ROC and channel length and the intracellular recording duration.
For both DRG neurons and HiPSC-CMs, there was no significant difference (P>0.05) in recording duration with ROC (Supplementary
Fig. 13 and Supplementary Table 4). Additionally, we observed that
increasing the channel lengths showed no difference in recording
duration in the DRG cells (P>0.05), while for the HiPSC-CMs
channel length increasing from 50 to 500nm for the 0.75µm ROC
and from 50 to 2,000nm for the 1.0 µm ROC U-NWFET resulted in
a statistically significant (P=0.007 and 0.027, respectively) increase
in recording duration (Supplementary Fig. 13 and Supplementary
Table 4). The lack of correlation between ROC and recording distribution suggests that loss of intracellular access is related to curvature-independent factors. We attribute the observed increase in
recording duration for longer channel length measurements on
HiPSC-CMs to the higher probability of maintaining a partially
internalized configuration during cell contraction-induced instabilities. Recordings obtained on DRG neurons have shorter duration than HiPSC-CMs, possibly reflecting the reported differences
in cell membrane mechanical properties as neuron membranes are
generally less fluidic than those of cardiac cells30.
We further ask whether deterministic fabrication with size
and geometry control could enable multisite intracellular recording within a single cell using two U-NWFETs on one probe arm,
recording from cell networks using independent U-NWFET
probes, and/or simultaneous measurement of intracellular/extracellular action potentials from a single cell by two U-NWFETs with
different ROC and channel lengths (Fig. 5a). First, a single DRG
neuron soma was brought into contact with a pair of U-NWFETs
1.6 1.8 2.0 2.2 2.4
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-530
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10 ms
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b
c
20
mV
20
mV
10 ms
i iii
0.8
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0 1 2 3 4 5 6
d
i
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2 mV
10 ms
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a
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i ii
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ii
Fig. 5 | Multiplexed electrophysiological recording by U-NWFET probes.
a, Schematics of simultaneous multisite intracellular recording from a single
neuron by paired U-NWFETs on one probe arm (i), multiplexed intracellular
recording from different cells by U-NWFETs on different probe arms
(ii) and simultaneous intracellular/extracellular recording from one cell
by paired U-NWFETs on one probe arm (iii). b, Simultaneous intracellular
recording from one DRG neuron by two ~50 nm FETs with 0.75 μm ROC on
one probe arm with a 2 μm separation (i); derivative of traces in the region
marked by a dashed box (ii). The vertical dashed guiding line in ii indicates
the time point of the first action potential. No time delay is observed.
c, Multiplexed intracellular recording from two HiPSC-CMs by one paired
U-NWFET probe (i) and one single U-NWFET probe (ii); derivative of the
marked region (iii). The two probes arms are fabricated with a distance of
350 μm between them. d, Simultaneous intracellular/extracellular recording
from one HiPSC-CM by one ~50 nm FET with 0.75 μm ROC (top red trace,
original intracellular signal; bottom red trace, derivative of intracellular
signal) (i) and one ~2,000 nm FET with 1.5 μm ROC on one probe arm with
2 μm separation (ii). (iii) Closer examination of the marked region.
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(each with an ~50nm channel and 0.75μm ROC) separated by 2μm
on one probe arm. The simultaneously recorded intracellular action
potential amplitudes (Fig. 5b(i)) exhibited values of 46 and 28mV
from the two U-NWFETs. The derivative of two action potentials
signals (Fig. 5b(ii)) and overlay of the two traces scaled to the
same peak amplitudes (Supplementary Fig. 14a,b) shows that
the peaks coincide with each other, indicating there is no discernible delay or waveform difference observed in the soma between
the two channels.
Second, a layer of cultured HiPSC-CMs was brought into contact with paired U-NWFETs on the same probe arm (2 µm separation) and a third single U-NWFET probe separated by 350µm from
the paired probe (all three U-NWFETs with ~50nm channel and
0.75μm ROC). The paired probe recorded the intracellular action
potential within one cell with action potential amplitudes of 54mV
and 47mV (Fig. 5c(i)) in the two channels, while the third probe
simultaneously recorded from another cell with an amplitude of
62mV (Fig. 5c(ii)). Comparison of the time derivatives (Fig. 5c(iii))
showed no discernible delay in the paired channels, while there was
~6ms delay between paired and single probes. This delay time and
probe separation yield a signal propagation speed of ~5.8 cms−1
,
which agrees with that reported in the literature31. An overlay of the
action potentials (Supplementary Fig. 14c,d) shows good agreement
in the rising phase, and small deviations in the repolarization phase
that can be attributed to different changes in the two U-NWFET/
cell junctions as a result of mechanical contraction32.
Third, paired U-NWFETs containing one ~50nm FET with
0.75μm ROC and one ~2,000nm FET with 1.5μm ROC on a single
probe arm with 2μm separation were fabricated and brought into
contact with a HiPSC-CM (Fig. 5d). The channel with the ~0.75µm
ROC and 50nm FET measured waveforms of ~50mV characteristic of intracellular cardiac action potentials (Fig. 5d(i)), while
the channel with the 2.0µm ROC and ~2,000nm FET measured
sharp downward spikes of ~2mV and <5ms duration characteristic of extracellular cardiac action potentials (Fig. 5d(ii)). Close
examination of the rising phase and derivative of the intracellular
trace in comparison to the extracellular trace (Fig. 5d(iii)) shows
that the measured extracellular signal is dominated by a downward
peak at the same time as the upwards phase of the action potential. Extracellular waveforms measured using metal electrodes show
increasing potential from capacitive coupling with the intracellular space, which should be aligned with the time derivative of the
intracellular signal28,33, followed by a decrease from the inward Na+
current during action potential firing33. Our observed monophasic
negative waveform suggests the small size of our recording element
decreases capacitive coupling with the interior of the cell so that
only local potential changes derived from inward Na+ currents are
observed. Furthermore, previous reports of simultaneous extracellular and intracellular measurement required separate patch-clamp
and metal electrode array recording systems, which can introduce
complications such as spatial mismatch of recording sites and difficulty in temporal synchronization28,33. In comparison, our multiplexed measurement strategy provides more localized information
to correlate the extracellular and intracellular action potentials.
Finally, we demonstrate the scalability of our U-NWFET devices
by fabricating six device regions containing 135 working single or
multi U-NWFET devices (out of 168 addressable device sites) on
a 76mm wafer (Supplementary Fig. 15a,b). For typical cell experiments, the wafer is subsequently ‘diced’ to yield six chips that are
individually mounted on printed circuit boards (Supplementary
Fig. 5a). Separate measurements from HiPSC-CMs for each of the
six chip device arrays yielded an intracellular signal from at least
eight devices for each chip (Supplementary Fig. 15c). In one device
array, we designed a series of probes containing 1–4 nanowires per
probe arm (Supplementary Fig. 16a,b), and show simultaneous
recording of action potentials in 10 channels from four separate
cells (Supplementary Fig. 16c). These experiments validate the
potential for using our U-NWFET probes for multiplexed recording to study the electrophysiology of cell networks.
Conclusions
In summary, we have demonstrated that the scalable ultrasmall
U-NWFET probe arrays fabricated using deterministic shapecontrolled nanowire assembly and selective spatially defined solidstate semiconductor-to-metal transformation have the capability
to record full amplitude intracellular action potentials from primary neurons and other electrogenic cells, and have the capacity
for multiplexed recordings. These new studies complement other
efforts from several groups focused on developing solid-state
nanodevices for cell electrophysiology. Although our number of
recording channels is limited compared to the hundreds of channels
demonstrated in electrode-array-based strategies12,14, it is notable
that our U-NWFETs show the capability to record full amplitude
intracellular actions potentials that are similar to patch-clamp
recordings, but now in a scalable format. This flexible device structure/fabrication approach has further provided direct information
about the relationship between recording amplitude as a function
of device curvature and size, and thus supports the developing
‘curvature hypothesis’ relating nanotopography to endocytosis and
cytoskeleton dynamics17. One key challenge, encountered both by
U-NWFETs and other nanodevices, is the long-term stability of the
intracellular recording. We hypothesize that future studies exploring either (1) chemical anchoring via surface functionalization with
groups that can bind to the actin cytoskeletal localized near highly
curved membranes17,18,25 or (2) physical anchoring using spicule
mesostructures34 and/or modulation of nanowire morphology29 to
increase the physical detachment force could improve the stability
of the intracellular device configuration. Supporting the potential
of these proposed directions, previous experiments indicate that
functionalized free-standing nanowires can remain in the interior
of neurons for at least several hours35. More generally, this deterministic nanowire-based fabrication strategy can be incorporated
into other platforms, such as free-standing probes16, which allow
precise targeting of individual cells or subcellular structures, and
mesh electronics36,37 for in vivo measurements.
Online content
Any methods, additional references, Nature Research reporting
summaries, source data, statements of code and data availability and
associated accession codes are available at https://doi.org/10.1038/
s41565-019-0478-y.
Received: 29 December 2018; Accepted: 15 May 2019;
Published: xx xx xxxx
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Acknowledgements
C.M.L. acknowledges support from the Air Force Office of Scientific Research (FA9550-
14-1-0136). S.S.Y. acknowledges an NSF Graduate Research Fellowship. This work was
performed in part at the Center for Nanoscale Systems (CNS) of Harvard University.
Author contributions
Y.Z., S.S.Y. and C.M.L. conceived and designed the experiments. Y.Z., S.S.Y. and A.Z.
performed the experiments and analysed the data. Y.Z., S.S.Y., A.Z. and C.M.L. co-wrote
the paper. All authors discussed the results and commented on the manuscript.
Competing interests
The authors declare no competing interests.
Additional information
Supplementary information is available for this paper at https://doi.org/10.1038/
s41565-019-0478-y.
Reprints and permissions information is available at www.nature.com/reprints.
Correspondence and requests for materials should be addressed to C.M.L.
Journal peer review information: Nature Nanotechnology thanks Bozhi Tian, Bruce
Wheeler and other anonymous reviewer(s) for their contribution to the peer review of
this work.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
© The Author(s), under exclusive licence to Springer Nature Limited 2019
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Methods
Nanowire synthesis. Si nanowires (p-type, 15nm diameter) were synthesized
using a gold nanocluster-catalysed vapour–liquid–solid growth method21.
Growth substrates (15×60mm2
pieces of Si wafer with 600nm thermal oxide,
Nova Electronic Materials) were oxygen plasma cleaned (100W, 2min, 50 cubic
centimetres per minute (s.c.c.m.) O2, PJ Plasma Surface Treatment System), treated
with poly-l-lysine solution (0.1%, 150,000–300,000 gmol−1
, Ted Pella) for 5min,
rinsed thoroughly with deionized (DI) water and dried with nitrogen. Ten, 1ml
of aqueous solution of 10nm gold nanoparticles (Ted Pella) with a concentration
of 1.9×1012particles per ml was dispersed38 on the substrate for 2min followed
by thorough rinsing with DI water and drying with nitrogen (gold nanoparticle
surface concentration, 0.01–0.04 particles per μm2
). Te substrate was then
placed into a home-built chemical vapour deposition reactor and the system was
evacuated to a base pressure of 0.6mtorr. Nanowires were synthesized at 430 °C at
a total pressure of 40 torr with gas fow rates of 2.5 s.c.c.m. silane (SiH4, 99.9999%,
Voltaix) as the silicon reactant, 3.1 s.c.c.m. diborane (B2H6, 100ppm in H2, Voltaix)
as the p-type dopant, and 60 s.c.c.m. hydrogen (H2, 99.999%, Matheson) as the
carrier gas. Typical growth times of 1h yielded nanowires with average
lengths of ~50µm.
U-NWFET probe array fabrication. Key steps involved in the fabrication of
U-NWFET probe arrays are shown in Fig. 2 and Supplementary Fig. 1, with the
key parameters as follows:
(1) LOR 3A (Microchem) and diluted S1805 (S1805: Tinner-P=1:2 (vol:vol),
Microchem) were spin-coated on a Si3N4/SiO2-coated Si wafer (200nm Si3N4,
100nm SiO2 on p-type Si, 0.005Ωcm, or 600nm thermal SiO2 on n-type Si,
0.005Ωcm, Nova Electronic Materials) and baked at 180 °C for 5min and
at 115 °C for 1min, respectively. Te photoresist was patterned by photolithography with a Maskless aligner (MLA150) and developed (MF-CD-26,
MicroChem Corp.) for 30 s. Following this photolithography process, a
60-nm-thick Ni sacrifcial layer was deposited by thermal evaporation (Sharon
Vacuum Co.), followed by a lifof step (Remover PG, MicroChem Corp.)
(Supplementary Fig. 1a). Te size of the Ni sacrifcial layer was designed to
accommodate the size of the U-NWFET probe: 30μm×100μm for single
U-NWFET probes (Supplementary Fig. 1h(i)) or 90μm×100μm for up to
four U-NWFETs probes per bend-up probe arm (Supplementary Fig. 1h(ii)).
(2) Te photolithography process in step 1 was repeated to defne an 80-μmlong bottom region for sputter deposition of a 60nm Si3N4 passivation layer
(Orion 3 Sputtering Systems, AJA International). Te main body of the Si3N4
passivation layer (75μm long) was deposited on the Ni sacrifcial layer with
5 μm Si3N4 extending outside of the sacrifcial region (Supplementary Fig. 1b).
(3) LOR 1A (Microchem) and diluted S1805 (S1805: Tinner-P=1:2 (vol:vol))
were spin-coated and baked at 180 °C for 5min and at 115 °C for 1min,
respectively. Te photolithography process in step 1 was repeated to defne
arrays of trenches with shapes and ROCs as described in the main text (Supplementary Fig. 1c).
(4) Te shape-controlled deterministic nanowire assembly was used to align
disordered straight nanowires into U-shaped arrays as described previously21 (Supplementary Fig. 1d). Briefy, a wafer with an array of trenches was
mounted onto a micromanipulator-controlled movable stage, covered with
mineral oil (viscosity v≈70mPa s, #330760, Sigma-Aldrich) as the lubricant,
and then the nanowire growth substrate was brought into contact with the
target substrate with controlled contact pressure. Te target substrate was
moved at a constant velocity of ~5mmmin–1 with respect to the fxed nanowire growth substrate; the growth substrate was then removed and the target
substrate rinsed with octane (98%, Sigma-Aldrich) to remove the lubricant.
Estimations of the U-shaped nanowire strain were calculated and are shown
in Supplementary Table 2.
(5) Al2O3 was deposited directly afer the nanowire assembly by atomic layer
deposition (S200, Cambridge NanoTech) with 1 cycle (1.4Å) at 80 °C to
fx the U-shaped nanowires on the bottom passivation layer and then all
photoresist was removed in Remover PG (Supplementary Fig. 1e).
(6) Step 1 was repeated to simultaneously pattern electrical interconnects to the
U-NWFET as well as connects to the large pads used as the input/output
(I/O) region. Native oxide on the nanowire was etched with a bufered oxide
etch (BOE, 7:1, Microchem) for 10 s before thermal deposition of asymmetrically strained metal Cr/Au/Cr (1.5/60/60nm). Te strained metal leads
the U-NWFET probe to bend of the wafer surface following etching of the
sacrifcial layer, like that described in previous work21.
(7) Step 2 was repeated to deposit 60nm of Si3N4 as electrical passivation
over exposed metal features except for the I/O pad region (Fig. 2b and
Supplementary Fig. 1f).
(8) Electron-beam lithography (EBL) or photolithography was used to defne
the Ni difusion region with shape and position as described in the main
text. Specifcally, EBL was used for U-NWFET probes with ~50nm (Fig. 2c)
and ~500nm (Supplementary Fig. 2c) channel length. For the EBL process,
copolymer MMA (EL6, Microchem) and polymethyl methacrylate (PMMA,
950-C2, Microchem) were spin-coated and baked at 180 °C for 5min,
sequentially. Te resists were then patterned with an EBL system (ELS-F125,
Elionix) and developed (MIBK/IPA 1:1, MicroChem Corp.) for 60 s. For
U-NWFET probe with ~2,000nm channel length (Supplementary Fig. 2d),
the same photolithography process in step 1 could be used to defne regions
for Ni deposition. Native oxide on the nanowire was removed by BOE for 10 s
before deposition of 20nm Ni via thermal evaporation. Afer lifof, the chip
was annealed using a Rapid Termal Processor (RTP, 600xp, Modular Process
Technology) in forming gas (H2:N2 10:90) at 350 °C for 5min to transform the
Si nanowire segments underneath and adjacent to the Ni difusion layer to
nickel silicide22, thereby generating a localized sensing element (Supplementary Fig. 1g).
(9) Polydimethylsiloxane (PDMS) was prepared by frst pouring Sylgard 184
(Dow Corning) elastomer (mixed in a 10:1 ratio of base to curing agent)
into a Petri dish, and then curing overnight at 55 °C in a convection oven.
A PDMS chamber with ~20×30mm2
opening and ~0.5 cm sidewalls was cut
from the cured PDMS and mounted around the device region using Kwik-Sil
silicone adhesive (World Precision Instruments). A printed circuit board
(PCB, UXCell) connector was then mounted adjacent to the I/O region of the
devices and wire-bonded to the U-NWFET probe I/O pads (Supplementary
Fig. 5a). Probes were kept in a Dry-Keeper desiccator cabinet (H-B Instrument-Bel-Art). Before electrical characterizations and/or electrophysiological
measurements, the Ni sacrifcial layers and remaining Ni from the difusion
layer were removed in nickel etchant (Nickel Etchant TFB, Transense) for
3–5min, which allowed release of these devices into a 3D bend-up structure
(Fig. 2d and Supplementary Fig. 1h). Following release, the devices were
rinsed in DI water 5–10 times for 20 s each.
Device characterization. Overview optical images of the measurement set-up and
U-NWFET probe chip to instrument I/O area were acquired with an SLR digital
camera (Canon), and higher-resolution bright-field optical images of individual
U-NWFET probes and probe arrays were acquired by an Olympus BX50WI system
with Andor Luca electron-multiplying charge-coupled device camera. Highresolution SEM images of nanowires, including intermediate fabrication steps, were
acquired using a Zeiss Ultra Plus field emission SEM (Carl Zeiss). A BSE detector
was used to obtain high-resolution composition-sensitive maps based on the
electron elastic scattering difference of atomic number on the sample. U-NWFETs
fabricated for characterization did not have a Ni sacrificial layer to improve
contrast during SEM imaging. The BSE images show the silicon and nickel silicide
segments on U-shaped nanowires: the bright region indicates nickel/nickel silicide
and the dark region indicates p-type Si.
For electrical characterization, one arm of the U-NWFET was considered as
the source, and the other arm is considered as the drain. Voltage Vds was applied
between the source and drain of the U-NWFET, and the resulting current, Ids,
was measured. The electrical conductance (Ids/Vds) of the U-NWFET devices was
measured in the dry state by sweeping Vds between −1 and 1V and measuring
Ids using a homemade battery-powered 16-channel current preamplifier with
bandwidth of 6 kHz, which amplified the current signal for recording using a
16-channel analog-to-digital converter (Axon Digidata 1440A, Molecular Devices)
controlled by pCLAMP 10.7 software (Molecular Devices). The Ids–Vds data were
recorded in an air/dry state.
Surface functionalization of U-NWFET probes. Phospholipid vesicles were
prepared for use in functionalized U-NWFET probes in the following manner,
similar to previous papers15,16. (1) 1,2-dimyristoyl-sn-glycero-3-phosphocholine
(DMPC, Avanti Polar Lipids) was suspended in chloroform (anhydrous, >99%,
Sigma-Aldrich) to a concentration of 20mgml−1
and mixed with 1% mass of
1-myristoyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-snglycero-3-phosphocholine (NBD-lipid, Avanti Polar Lipids). (2) The solution
of DMPC/NBD-lipid was then placed into a vacuum desiccator for at least 6h
to evaporate off the chloroform. (3) The resulting powder was resuspended in
phosphate buffered saline (1× PBS, HyClone) to a concentration of 1mgml−1
and the lipid solution was placed in a water bath at 37 °C for at least 2h with
periodic agitation using a vortex mixer (30 s every 20min, Maxi Mix II, Barnstead/
Thermolyne Corp.) to ensure full rehydration. (4) The resulting lipid solution was
sonicated using a tip sonicator (25% amplitude, 10 s/15 s pulse on/off, Branson
Ultrasonics Sonifier S-450l, Branson Ultrasonics) at ~37 °C for 2h. (5) Following
sonication, the lipid solution was sterile filtered (0.2 µm Acrodisc syringe filter,
PN 4192, Pall Corp.) and used within 1h of preparation.
Immediately before measurements, U-NWFET probe arrays with a mounted
PDMS chamber (Supplementary Fig. 5b) were incubated for 2h in 1.5ml of the
prepared lipid vesicle solution to allow functionalization of U-NWFET as reported
previously for other nanowire devices15,16. Following incubation, the U-NWFET
probe arrays were rinsed in Tyrode’s solution (in mM: NaCl 155, KCl 3.5, MgCl2 1,
CaCl2 1.5, HEPES 10, d-glucose 10, pH7.4 for DRG neurons, or NaCl 138, KCl 4,
CaCl2 2, MgCl2 1, Na2HPO4 0.33, HEPES 10, glucose 10, pH7.4 for HiPSC-CMs, all
chemicals in Tyrode’s solution were purchased from Sigma-Aldrich) 5–10 times for
20 s, ~3ml each.
Device characterization in Tyrode’s solution. Following phospholipid
modification, electrical measurements were carried out in Tyrode’s solution.
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The electrical conductance of the U-NWFETs was continuously measured by
recording the drain–source current (Ids) at a fixed source–drain d.c. bias between
0.1 and 0.2V by the electronic measurement set-up mentioned above. The
sensitivities (transconductance) were then obtained by sweeping an Ag/AgCl
reference electrode (2.0×4.0mm, E-201, Warner Instruments) between −100mV
and 100mV and measuring the corresponding linear change in U-NWFET
conductance. The measured average (in 10 samples) conductance of U-NWFETs
for channel lengths of ~50nm, ~500nm and ~2,000nm are 3.3±0.6, 0.7±0.2 and
0.3±0.1μS, with average sensitivities of 5.4±1.3, 2.3±0.7 and 0.9±0.3μSV−1
respectively. An inverse relationship exists between conductance, and consequently
transconductance, and channel length, as expected from the relationship:
(G=σA/L), where G is the channel conductance, σ is the electrical conductivity,
A is the cross-sectional area of the wire and L is the channel length39.
To estimate the noise level of the U-NWFETs devices, the conductance of the
10 devices for each channel length was measured using the Ag/AgCl reference
electrode to fix the solution voltage at 0 for ~5 s. The standard deviation of the
measured conductance was used to obtain the noise for each device in μS. Then,
the transconductance of each device was used to convert the measured noise in
μS into a value in mV, and the resulting number was multiplied by 3 to estimate
the limit of detection as per convention. Averaging the 10 values for the limit of
detection (in mV) for each channel length resulted in noise levels of 0.90±0.60,
1.2±0.9 and 1.9±0.9mV for the ~50, 500 and 2,000nm devices, respectively.
Preparation of flexible cell culture substrates. A master mould for the
culture substrate was first prepared by spin-coating SU-8 2000.5 (Microchem)
onto a Si wafer and patterning repeating 3-μm-wide lines with 3μm spacing
using photolithography. After patterning, the master mould was hard baked
on a hot plate at 180 °C for 2h, and then silanized with tridecafluoro-1,1,2,2-
tetrahydrooctyl-1-trichlorosilane (Sigma-Aldrich) for 2h in a vacuum desiccator,
to enhance release of the PDMS template from the master mould40.
Flexible PDMS cell culture substrates were prepared by spin-coating Sylgard
184 elastomer mixed in a 10:1 ratio of base to curing agent onto the master mould
at 250 r.p.m. for 1min. The PDMS on the master mould was then cured in a
convection oven set to 180 °C for 2h, resulting in a thickness of ~220μm, and cut
into pieces (~10×10mm2
) for cell culture. Before cell culture, the PDMS substrates
were autoclaved at 125 °C for 1h, treated by O2 plasma (100W, 2min, 50 s.c.c.m.
O2) and then washed in a 75% (vol/vol) solution of ethanol (200 proof, KOPTEC)/
water for 1h.
For DRG neuron culture, the PDMS was first functionalized with 40µgml−1
poly-d-lysine (molecular weight of >300,000 gmol−1
, Sigma-Aldrich) in DI water
for 1h at room temperature. After poly-d-lysine functionalization, the PDMS
was washed twice in DI water for 30 s each and air dried, and then functionalized
with 20 µgml−1
laminin (Thermo Fisher Scientific) in Leibovitz’s L-15 (Thermo
Fisher Scientific) for 1h at room temperature. Laminin solution was removed
immediately before the cell suspension was plated on the PDMS.
For HiPSC-CM culture, the PDMS was functionalized sequentially with
(1) 1% (3-aminopropyl)triethoxysilane (Sigma-Aldrich) in a 95% (vol/vol) solution
of ethanol/DI water for 20min at room temperature, followed by washing three
times in ethanol for 30 s each, and three times in DI water for 30 s each; (2) 2.5%
(vol/vol) glutaraldehyde (grade I, 50% in H2O, Sigma-Aldrich)/water for 1h at
room temperature, followed by washing three times in DI water for 30 s each;
(3) Geltrex matrix (Thermo Fisher Scientific) at 37°C for ~8h. The Geltrex solution
was removed immediately before the cell suspension was plated onto the PDMS.
Cell culture. Dissociated DRG cells were prepared as described previously35 and
cultured in the CO2 incubator overnight before use. Cells that can spontaneously
fire (Supplementary Fig. 8) were selected for recording. HiPSC-CMs were
cultured as described in the NCardia online protocol41. Cryogenically frozen
Cor.4U HiPSC-CM vials (Cor.4U>250k cells, Ncardia Group) were thawed in a
37 °C water bath, and 0.5ml of proprietary Cor.4U cell medium (Ncardia Group)
preheated to 37 °C was added to the vial. The cell solution was then homogenized
by gentle aspiration and seeded at 75,000 cells per cm2
to achieve confluency
onto the prepared PDMS substrates. Immediately following cell seeding, the cell
culture was left at room temperature for 20min to allow the solution to settle and
ensure an even distribution of cells. The cells were then cultured in a 5% CO2,
37 °C incubator and the Cor.4U cell medium was changed 6h following plating.
Subsequently, the medium was changed every day and the cells were used within
2 weeks following seeding, once a uniformly contracting layer was observed.
Electrophysiological recording with U-NWFET. The Ag/AgCl reference
electrode was used to fix the extracellular Tyrode’s solution voltage to 0V for cell
measurements. A PDMS sheet with cultured DRG neurons or HiPSC-CMs was
fixed upside down onto a homebuilt vacuum wand mounted on a 40nm step
resolution x–y–z micromanipulator (MP-285, Shutter Instruments) connected to a
micromanipulator controller (MPC-200/ROE-200, Sutter Instruments) to position
the cells over and bring the cells into contact with the U-NWFETs (Supplementary
Fig. 5c). The Tyrode’s solution was maintained at room temperature for the DRG
neuron experiments and at ~37 °C for the HiPSC-CM experiments. For longer
(>3min) HiPSC-CM intracellular recording (Supplementary Fig. 9b,d), high pass
filters were set to 0.4Hz, similar to the approach used by other groups12,42,43.
Patch-clamp recording. Patch-clamp recording was performed at room
temperature using a Multiclamp 700B amplifier (Molecular Devices) and a
Digidata 1440A Digitizer Acquisition System, controlled by pCLAMP 10.7
software (Molecular Devices). Micropipettes were prepared using a micropipette
puller (P-97, Sutter Instruments) and the pipette tip resistance ranged between
5 and 10MΩ. DRG neurons were cultured on a glass coverslip with the same
modification as PDMS. Recording from DRG neurons was carried out in Tyrode’s
solution. The micropipettes were filled with an internal solution consisting of
(in mM): potassium l-aspartate 140, NaCl 13.5, MgCl2 1.8, ethylene glycolbis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) 0.09, HEPES 9,
phosphocreatine di(tris) salt 14, adenosine 5′-triphosphate (ATP) magnesium salt
4, guanosine 5′-triphosphate (GTP) tris buffered 0.3, pH7.2 adjusted with KOH6
.
All chemicals in the internal solution were purchased from Sigma-Aldrich,
except GTP tris buffer, which was purchased from Thermo Fisher.
Data availability
The data that support the findings of this study are available from the
corresponding author upon reasonable request.
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