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Nano Letters


Figure 1. (A) Scanning electron microscopy (SEM) image of a 3D 64-electrode array (scale bar 500 μm); (B) nanopillar electrodes with guide lines
on the passivation layer (scale bar 20 μm); (C) silicon substrate with pattern of gold nanopillars for guidance tests (scale bar 200 μm); (D) single
square design with nodes connected by 200 μm lines (scale bar 50 μm).

length and the width of the pattern. As mentioned before, 2D
guidance is driven by a receptor-mediated mechanism based on
extracellular matrix recognition; it was proposed in the recent
years to additionally create micro-nano patterning on substrates
to induce cell proliferation, alignment, and stretching.30−32
In this Letter we report about the possibility to use 3D
nanostructures for coupling cardiomyocyte-like cells with our
recording device. In particular, we investigated how one can
combine cell guidance and extracellular recordings of cardiac
cells on a chip at the same time. Therefore, we studied the
effect of cell guidance on mushroom-shaped 3D gold
nanostructures which were fabricated onto a MEA chip for
extracellular recordings.
In this work, we used a MEA processed as shown in ref 33.
The electrode layout was adapted from ref 34, while we
increased the overall chip size to 24 × 24 mm2 to simplify the
flip-chip encapsulation to a top contact chip. For the fabrication
of the gold nanostructures we refer to ref 23. Briefly, a thin gold
film was deposited on the substrate, which was then covered by
a poly(methyl methacrylate) (PMMA) e-beam resist (Allresist
GmbH, Berlin, Germany) with a thickness of about 1 μm. The
apertures around 500 nm have been exposed by means of ebeam lithography. Consequently, the openings were filled by
electroplated gold using the sputtered gold film as a
background electrode. The gold nanostructures were electroplated on top of the planar MEA gold electrode with a diameter
of 8 μm as shown in Figure 1A−B. Additionally, gold lines with
a length of 180 μm have been fabricated between adjacent
electrodes on the top of the passivation layer without shortcircuiting the electrodes (Figure 1B). The width of the lines
was about 15 μm in the case of two parallel lines of pillar
featuring a 10 μm pitch and was scaled up to six parallel lines
when the smallest pitch was fabricated. In the next processing
step all of the gold strip-lines were electrically short-circuited by
a 50 nm aluminum layer. This circuit was electrically connected
to the MEA electrodes to galvanize simultaneously the cell
guiding mushroom lines and the electrode gold nanostructures

of the MEA itself. Finally, the resist was removed, and the
aluminum shorts were etched with an aluminum wet etchant
(ANPE80/5/5/10, Microresist Technology, Berlin, Germany).
Adopting these parameters, we were able to fabricate
mushrooms with a diameter of about 500 nm, a stalk height
of about 1 μm, and a cap height of 200 nm. The overall
effective surface area of an individual pillar was about 4.5−10
μm2, depending on the surface roughness of the cap, resulting
in a total electrode surface of about 77−115 μm2 in case of the
8 μm (diameter) electrodes for electrophysiological measurements. We determined the equivalent capacitance values of
about 30 pF per electrode with the help of impedance
spectroscopy. In addition to the MEAs with cell-guiding
nanostructures, we fabricated 64 gold pillar node containing
samples on silicon oxide for the optimization of spine
interspacing in cell guide lines (Figure 1C−D). The pitch
between nanopillars was varied in the range from 2 to 10 μm.
To cultivate cardiomyocyte-like cell line HL-1,35 the Si test
samples and the MEAs were cleaned in flowing ultrapure water
for 2 h and then sterilized for 30 min with UV light. After the
sterilization, the surface of the substrates was coated with
fibronectin at a concentration of 1 mL in 200 μL of 0.02%
Bacto TM Gelatin (Fisher Scientific) for an incubation time of
1 h. Confluent HL-1 cells in a T-25 flask were treated with
0.025% trypsin/EDTA, suspended in 5 mL of Claycomb
medium, and centrifuged for 5 min at 1700 rpm. The pellet was
then resuspended in 3 mL of medium, and 30 μL was plated on
the substrates. After 15 min 1 mL of medium was added, and
the substrates were incubated for 3 days until the cells formed a
confluent monolayer on the lines and started to contract. The
living cells on the silicon substrates were stained with 1 mM
calcein AM (Invitrogen) and 1 mM ethidium homodimer in
phosphate-buffered saline (PBS) solution (137 mM NaCl, 2.7
mM KCl, 8 mM Na2HPO4, 1.8 mM KH2PO4), and the image
acquisition was performed using an Axio Imager Z.1 (Carl Zeiss
AG, Oberkochen, Germany).
B | Nano Lett. XXXX, XXX, XXX−XXX