than 1 μV rms with a dynamic range of ±10 V (after
ampliﬁcation). For cellular measurements we limited the
eﬀective bandwidth with a high pass ﬁlter (AC coupling) and
a low pass ﬁlter (high frequency cutoﬀ) from 1 Hz to 3 kHz.
The analysis of the data was performed with a script written in
MATLAB (Mathworks, Natick, USA) and ORIGIN (OriginLab, Northampton, USA).
The spontaneous activity of the HL-1 was recorded by the
3D nanostructured MEAs with a nominal electrode footprint of
8 μm diameter with mushroom nanopillars, and on the guiding
lines we fabricated nanopillars of a 4 μm pitch according to the
guidance results. The traces of spontaneous action potentials
are shown in Figure 5A, showing the voltage recordings of four
electrodes as a function of time. The frequency of the action
potentials amounted 0.62 ± 0.15 Hz for every trace of the
example. Due to the peculiar signal propagation of the
cardiomyocytes, it is possible to determine a signal propagation
speed of 28.6 mm/s. Considering an interval of 1−2 s (Figure
5B), one can calculate the delay between an action potential on
one electrode and another electrode serving as a reference in
To chemically stimulate the cells, norepinephrine was added
to cell media with a concentration of 1:1000. Norepinephrine
eﬀects an increase of the contraction rate of the cardiomyocytes
(Figure 6), resulting in a frequency of about 0.89 ± 0.17 Hz in
have more than a 90% eﬀective guidance eﬀect on HL-1 cells.
Moreover, we were able to investigate the cell response to the
pillar interspace with focused ion beam cross sectioning and
scanning electron microscopy. These guidance results represent
an improvement compared to results obtained with other
techniques previously mentioned and in addition the 3D
nanostructure-based guidance represents a novel technique for
cell patterning. The mushroom-like structures are furthermore
suitable for extracellular recordings of action potentials and
combine eﬀective cell guidance with electrophysiological
investigations for cardiomyocyte-like cells. To the knowledge
of the authors, this is one of the very few examples, potentially
the ﬁrst, shown in the literature where a deﬁned nanostructure
was used eﬀectively and simultaneously for cell patterning and
*E-mail: a.oﬀenhaeusser@fz-juelich.de. Address: Leo Brandt
Strasse 1, 52428, Jülich, Germany. Tel. Oﬃce: +492461612330. Fax: +49246161-8733.
The authors declare no competing ﬁnancial interest.
We would like to acknowledge Marco Banzet for the help in the
fabrication process. Unless otherwise noted, all the chemicals
were supplied by Sigma Aldrich GmbH, Seelze, Germany.
(1) Thomas, C. A., Jr.; Springer, P. A.; Loeb, G. E.; Berwald-Netter,
Y.; Okun, L. M. Exp. Cell Res. 1972, 74, 61−66.
(2) Gross, G. W.; Rieske, E.; Kreutzberg, G. W.; Meyer, A. Neurosci.
Lett. 1977, 6, 101−105.
(3) Pine, J. J. Neurosci. Methods 1980, 2, 19−31.
(4) Choi, D. S.; Fung, A. O.; Moon, H.; Villareal, G.; Chen, Y.; Ho,
D.; Presser, N.; Stupian, G.; Leung, M. J. Nanosci. Nanotechnol. 2009,
(5) Kim, J.-H.; Kang, G.; Nam, Y.; Choi, Y.-K. Nanotechnology 2010,
(6) Wesche, M.; Hüske, M.; Yakushenko, A.; Brüggemann, D.;
Mayer, D.; Offenhäusser, A. Nanotechnology 2012, 23, 495303.
(7) Wang, K.; Fishman, H. A.; Dai, H.; Harris, J. S. Nano Lett. 2006,
(8) Yakushenko, A.; Gong, Z.; Maybeck, V.; Hofmann, B.; Gu, E.;
Dawson, M.; Offenhäusser, A.; Wolfrum, B. J. Biomed. Opt. 2013, 18,
(9) Verma, P.; Melosh, N. A. Appl. Phys. Lett. 2010, 97, 033704−
(10) Weis, R.; Fromherz, P. Phys. Rev. E 1997, 55, 877−889.
(11) Spira, M. E.; Hai, A. Nat. Nanotechnol. 2013, 8, 83−94.
(12) Brüggemann, D.; Wolfrum, B.; Maybeck, V.; Mourzina, Y.;
Jansen, M.; Offenhäusser, A. Nanotechnology 2011, 22, 265104.
(13) Keefer, E. W.; Botterman, B. R.; Romero, M. I.; Rossi, A. F.;
Gross, G. W. Nat. Nanotechnol. 2008, 3, 434−439.
(14) Gabay, T.; Ben-David, M.; Kalifa, I.; Sorkin, R.; Abrams, Z. R.;
Ben-Jacob, E.; Hanein, Y. Nanotechnology 2007, 18, 035201.
(15) Hai, A.; Shappir, J.; Spira, M. E. Nat. Methods 2010, 7, 200−202.
(16) Fendyur, A.; Spira, M. E. Front. Neuroeng. 2012, 5, 10.3389/
(17) Duan, X.; Gao, R.; Xie, P.; Cohen-Karni, T.; Qing, Q.; Choe, H.
S.; Tian, B.; Jiang, X.; Lieber, C. M. Nat. Nanotechnol. 2012, 7, 174−
(18) Hai, A.; Shappir, J.; Spira, M. E. J. Neurophysiol. 2010, 104, 559−
Figure 6. Action potentials recorded by 3D electrodes after chemical
stimulation with noradrenaline during a time frame of 12 s.
our experiment. Earlier works demonstrated the application of
nanostructured MEAs to be suitable for extracellular recordings
from HL-1 cells in the millivolt regime.6,12 On the other hand,
nanostructured MEA electrodes were successfully employed for
the recording of action potentials in the 10 mV range from
neuronal cell cultures.15 We show here the coupling of
cardiomyocytes on nanostructured electrodes. The recorded
signals from our gold nanopillars reached about 180 μV (peakto-peak) on some electrodes and were clearly distinguishable
from the background electrode noise. Xie et al.41 also measured
with their array of nanopillars (height 1.5 μm, diameter 150
nm) spontaneous activity from HL-1 cells in the range of about
100 μV (peak-to-peak) which is comparable with our results.
In conclusion, our 3D gold mushroom-shaped nanostructures were shown to be an appropriate tool for chip-based
simultaneous guidance and recording experiments. We were
able to determine that nanopillars with a pitch from 3 to 7 μm
dx.doi.org/10.1021/nl402901y | Nano Lett. XXXX, XXX, XXX−XXX