108 Int. J. Nano and Biomaterials, Vol. 4, No.

2, 2012

Adhesion and survival of electrogenic cells on gold

nanopillar array electrodes

Dorothea Brüggemann*
PGI-8/ICS-8,
Forschungszentrum Jülich GmbH,
Jülich, Germany
and
JARA – Fundamentals of Future Information Technology,
52425 Jülich, Germany
and
CRANN – The Naughton Institute,
School of Physics,
Trinity College Dublin,
Dublin 2, Ireland
E-mail: brueggemann@physik.rwth-aachen.de
*Corresponding author

Kristin E. Michael
Deceased; formerly of: PGI-8/ICS-8,
Forschungszentrum Jülich GmbH,
Jülich, Germany
and
JARA – Fundamentals of Future Information Technology,
52425 Jülich, Germany

Bernhard Wolfrum and Andreas Offenhäusser

PGI-8/ICS-8,
Forschungszentrum Jülich GmbH,
Jülich, Germany
and
JARA – Fundamentals of Future Information Technology,
52425 Jülich, Germany
E-mail: b.wolfrum@fz-juelich.de
E-mail: a.offenhaeusser@fz-juelich.de

Abstract: Cell-electrode interfaces play a critical role in extracellular

recording. Enlarging the electrode surface area with nanostructures yields
higher signal-to-noise-ratios due to lower interface impedance. Adhesion and
viability of various cell types on large-scale gold nanopillar electrodes to
improve cell-electrode coupling were investigated. Cardiac muscle and human
embryonic kidney cells survived and adhered well on gold nanopillars. The
muscle cells even protruded into inter-pillar cavities with diameters below
100 nm. However, an unexpectedly low viability and adhesion of primary rat

Copyright © 2012 Inderscience Enterprises Ltd.

 Adhesion and survival of electrogenic cells 109

neurons was observed on nanopillars. A cross-sectional analysis of the

cell-nanopillar interface showed large distances between neuronal cell bodies
and nanopillars, whereas the neurites adhered tightly. Furthermore, actin
assembly within the neuronal growth cones was modified on nanopillars. In
summary, the adhesion response of the investigated cell lines will be beneficial
for improved extracellular signalling, whereas a better understanding of
neuronal responses to nanotopographies is required to enhance the neuronal
viability.

Keywords: biosensor; nanostructured electrode; anodic alumina template; gold

nanopillars; cell adhesion and viability; neuronal cells and networks;
nanotopography; cell-electrode interface; biomaterial.

Reference to this paper should be made as follows: Brüggemann, D.,

Michael, K.E., Wolfrum, B. and Offenhäusser, A. (2012) ‘Adhesion and
survival of electrogenic cells on gold nanopillar array electrodes’, Int. J. Nano
and Biomaterials, Vol. 4, No. 2, pp.108–127.

Biographical notes: Dorothea Brüggemann received her PhD from the

Forschungszentrum Jülich on novel nanostructured gold electrodes for
improved extracellular signalling. Currently, she is a postdoctoral researcher at
Trinity College Dublin, Ireland where she works in the field of single molecule
manipulation using optical tweezers.

Kristin E. Michael sadly died shortly before the completion of this research
article. Until her death she worked as a postdoctoral researcher at the
Forschungszentrum Jülich. Her research interest was the enhancement of
neuronal adhesion and survival at cell-device interfaces for biosensing
applications.

Bernhard Wolfrum is the leader of the Helmholtz Young Investigator Group for
chip-based communication with cells at the Forschungszentrum Jülich. His
research interests are the electrochemical detection of redox-active molecules
such as neurotransmitters and the nanoscaled cell-chip interface.

Andreas Offenhäusser is a Director at the Institute of Complex Systems and the

Peter Grünberg Institute at the Forschungszentrum Jülich and jointly appointed
Professor for Experimental Physics at RWTH Aachen University. His main
research areas are the development of novel biosensors and studies on signal
processing in neuronal networks as well as on the biofunctionalisation of
surfaces.

This paper is a revised and expanded version of a paper entitled ‘Cell

viability and adhesion on nanostructured gold electrodes’ presented at the 17th
Annual Conference of the Section of Bioengineering of the Royal Academy of
Medicine in Ireland, Galway, 28–29 January 2011.

1 Introduction

Understanding signal propagation in cellular networks is important to the development of

neural prostheses, drug delivery systems, and biosensors. The electrical activity of
electrogenic cells can be recorded with planar microelectrode arrays (MEAs). Thereby,
110 D. Brüggemann et al.

the cell-chip coupling depends on the impedance of the electronic interface and the cell
adhesion. A tight cell-chip coupling is crucial to detect small amplitude signals due to an
increased voltage at the cell-substrate interface (Egert et al., 1998; Fromherz et al., 1991;
Heuschkel et al., 2002; Joye et al., 2009; Taketani and Baudry, 2006). Thus, the resulting
signal-to-noise ratio is strongly affected by the cell-electrode interface and the
electrode geometry (Isik et al., 2005). Often, the cell-electrode distance is too high
(Rutten, 2002) or the electrode is blocked by glia or dead cells yielding bad signal quality
(Heuschkel et al., 2002). Thus, for improved signal recording the interaction between
physically or chemically modified surfaces and different cell types is of major
importance.
In earlier fundamental studies the interface between diverse cell types and various
microstructured materials has already been investigated (Craighead et al., 1998; Curtis
and Wilkinson, 1997, 1998). With emerging possibilities in the field of nanotechnology
these studies were extended to nanotopography-cell interfaces, such as silicon or quartz
groove-ridge topographies, PMMA, PLGA and PDMS nanopatterns, PLLA nanopits or
silicon pillars (Dowell-Mesfin et al., 2004; Flemming et al., 1999; Hwang et al., 2010;
Lim and Donahue, 2007; Loesberg et al., 2007; Martinez et al., 2009; Nealey et al., 2003;
Teixeira et al., 2006; Yim et al., 2005).
For the modification of MEAs three-dimensional tip microelectrodes have been
designed to avoid glia or dead cells on the electrodes (Heuschkel et al., 2002; Isik et al.,
2005; Nam et al., 2006; Thiébaud et al., 1997). To enable coupling to single cells in
networks many nanostructured electrode materials have been introduced recently. Meshes
and sheets of carbon nanotubes (CNTs) (Cellot et al., 2009; Galvan-Garcia et al., 2007;
Liopo et al., 2006; Lovat et al., 2005; Mazzatenta et al., 2007), vertically aligned
CNTs (Nguyen-Vu et al., 2006; Wang et al., 2006; Yu et al., 2007) and CNT coatings
(Gabay et al., 2007; Keefer et al., 2008) as well as nanoporous silicon (Moxon et al.,
2007) were employed for improved cell-electrode interfaces and increased neuronal
network activity. Metallic nanorods from platinum (Choi et al., 2009) and gold (Zhou
et al., 2009) as well as gold nanopillars (Brüggemann et al., 2011; Schröper et al., 2008)
were used as nano-modifications to improve cell-electrode coupling and to lower the
electrode impedance.
Recently, micron-sized cavities on gold microelectrodes were reported to improve
the coupling to cardiac muscle cells by bringing them to grow into the cavities (Hofmann
et al., 2011). Very good cell coupling and dense neuronal networks were also observed
with gold nanoflakes (Kim et al., 2010) and spine-shaped gold protrusions (Hai et al.,
2009a, Hai et al., 2009b, Panaitov et al., 2011). The protrusions were closely engulfed by
neurons, thus leading to high field potentials, which even allowed quasi-in-cell
recordings (Hai et al., 2010). Arrays of silicon nanowires (SiNWs) also supported
cell growth and adhesion (Qi et al., 2009) and penetrated individual cells naturally
(Kim et al., 2007). The idea of non-destructively incorporating nanostructures into cells
was recently implemented with vertically aligned carbon nanofibres (McKnight et al.,
2003, 2004), CNTs (Cai et al., 2005; Lacerda et al., 2007), SiNW arrays (Shalek et al.,
2010) and artificial stealth probes (Almquist and Melosh, 2010; Verma and Melosh,
2010).
Besides their use in biosensing applications, nanostructures have become attractive
for biomaterial modifications, e.g., in implant surfaces or tissue engineering. Patterns of
GaP nanowires (Prinz et al., 2008) and nano-imprinted patterns in PMMA substrates
(Johansson et al., 2006) were employed to control axonal outgrowth. Native and
 Adhesion and survival of electrogenic cells 111

carbon-coated TiO2 nanotube surfaces helped to accelerate osteoblast cell growth

(Brammer et al., 2011; Oh et al., 2006; Popat et al., 2007) and to enhance the
proliferation of endothelial cells (Peng et al., 2009). The strong adhesion to the TiO2
nanotubes was supported by filopodia protruding into the nanotubular architecture.
However, with vascular smooth muscle cells on TiO2 nanotubes a restriction of the cell
size was observed. Implants modified with ZnO nanorods were even adhesion resistant
and led to the death of fibroblasts and endothelial cells due to the lack of initial cell
spreading (Lee et al., 2008). These versatile studies on cell adhesion and proliferation on
various nanostructures indicate that further investigation of the cell-nanostructure
interfaces is necessary for understanding the underlying interactions in general and to
improve future biosensing applications in particular.
To optimise extracellular signal recording with gold nanopillar MEAs, we study here
the viability and adhesion of different electrogenic cells on large-scale gold nanopillar
electrodes and the resulting cell-nanostructure interface using live-dead staining,
scanning electron microscopy (SEM) and focused ion beam (FIB) cross-sections as well
as actin filament staining.

2 Experimental

2.1 Fabrication of gold nanopillar arrays and SEM analysis

The production of gold nanopillar arrays was carried out using a template-assisted
approach with nanoporous alumina membranes. For the sample fabrication, p-doped
silicon wafers with a (100) orientation (Si-Mat, Landsberg, Germany) were used as
substrates. The wafers were oxidised under wet conditions at 950°C in a diffusion
furnace (type TS-6304, Tempress Systems, Vaassen, Netherlands) to obtain a top layer of
1,500 nm SiO2. Afterwards, the wafers were covered sequentially with e-beam
evaporated thin films of titanium (10 nm), gold (200 nm) from a Balzers system (type
PLS 500, Liechtenstein) and thermally evaporated aluminium (800 nm, Leybold LH 560,
Cologne, Germany). As references, planar gold layers were prepared by e-beam
evaporation of titanium (10 nm) and gold (200 nm) onto a silicon wafer. Each cell culture
chip had a total size of 11 × 11 mm.
The aluminium top layer of the samples was anodised in an etching bath with oxalic
acid (0.3 M, Merck KGaA, Darmstadt, Germany) at 40 V. During the anodisation
self-assembled nanoporous alumina was formed. The thin remaining barrier layer of
aluminium oxide at the pore bottom was removed via etching in phosphoric acid
(5%, Merck KGaA) for approximately 40 minutes. During this time, the pores also
widened, connecting the nanochannels in the alumina template to the underlying
conducting gold film. The following electrochemical deposition was performed in the
galvanisation solution Pur-A-Gold from Enthone GmbH (Langenfeld, Germany) with
K[Au(CN)2] at a final gold concentration of 10 g/l and a pH value of 5.8 (measured at
60°C). Electrodeposition was carried out at 60°C with a current density of approximately
6 mA/cm2. To obtain nanopillars with an average height of 400 nm, the galvanisation
time was between 120 and 130 s. Finally, the alumina matrix was removed by soaking
the samples in KOH (20%, Merck KGaA) for one hour. This step yielded freestanding
gold nanopillar arrays in the inner part of the chip in a circular area of 0.35 mm2. For
process-related reasons the inner nanopillar areas were surrounded by planar gold at the
112 D. Brüggemann et al.

chip edges. The pillar geometry was characterised via SEM using a Gemini 1550 VP
device (Carl Zeiss, Jena, Germany). For each pillar array sample three SEM images of
the pillars in top view at 0° and in side-view at 60° were taken. Afterwards, the
geometrical structure of the arrays was evaluated with regard to pillar diameter and
height as well as the distance between the pillars. Finally, 33 pillar array samples with
similar geometries were chosen for the following cell culture experiments (average pillar
height ~300 to 400 nm, diameter ~60 nm and inter-pillar distance ~25 nm. A detailed
statistical analysis of the pillar diameters and inter-pillar distances was presented by
Schröper et al. (2008).

2.2 Surface modifications and cells

Before the respective coating procedures, the samples were cleaned in a flow cell with
ultrapure water from a Millipore MilliQ system for approximately five hours. The
samples were transferred to an ultrasonic bath for three minutes at intervals of 60 minutes
to remove eventual residues of chemicals from the nanostructuring process. After drying
with nitrogen, the samples were cleaned in oxygen plasma (type Femto, Diener electronic
GmbH and Co. KG, Nagold) for ten minutes to remove organic substances from the
surface. All cells were cultivated on gold nanopillar arrays and on planar gold surfaces as
control substrates.
Two electrogenic cell lines were investigated in these experiments: Human
embryonic kidney cells with transfected ion channels [HEK 293 Nav 1.4, kindly
provided by S.H. Heinemann (Chen et al., 2000)] and HL-1 cardiomyocytes (Claycomb
et al., 1998). The nanostructured and planar gold substrates were transferred to the sterile
bench and sterilised with UV light for one hour. For experiments with HL-1 cells the
surfaces were coated with fibronectin (Sigma Aldrich, Steinheim, Germany) at a
concentration of 5 μg/ml in 0.02% Bacto© Gelatin. The samples were incubated in
this solution for 1 hour at room temperature and rinsed in phosphate buffered saline
solution (PBS, Sigma) before HL-1 cells were seeded at approximately 70 cells/mm2 in
Claycomb Medium. The experiments with HEK cells were carried out with a coating of
20 μg/ml poly-L-lysine (PLL, Sigma) in Hank’s buffered salt solution (HBSS, Invitrogen
GmbH, Oregon). The substrates were incubated in the PLL solution for one hour and
washed with HBSS prior to cell seeding. HEK cells were then seeded with approximately
80 cells/mm2 and maintained in minimal essential medium (M2279, Sigma),
supplemented with 2 mM glutamine (Sigma), 1% non-essential amino acids (Invitrogen),
10% (v/v) fetal calf serum (Invitrogen), 100 units/ml penicillin (Invitrogen) and 0.1
mg/ml streptomycin (Invitrogen). Both HL-1 and HEK cells were incubated at 37°C in
5% CO2.
For the cell culture with cortical rat neurons, the gold nanopillar arrays were coated
with two different types of alkanethiol self-assembled monolayers supplied from
ProChimia (Sopot, Poland). The first SAM consisted of 1 mM HS(CH2)11NH2 in 100%
ethanol (absolute, Merck KGaA) and will hence be abbreviated by NH2. The second
solution consisted of mixed monolayers [1 mM of HS(CH2)11EG3 and 1 mM of
HS(CH2)11EG6NH2 in 100% ethanol], referred to as MML. The samples were incubated
in the respective SAM solutions for four hours and then rinsed with 100% ethanol.
Afterwards, they were sterilised in 70% ethanol, and the PEG chains were orientated in
sterile bi-distilled water. Samples were placed in 35 mm dishes coated with a background
 Adhesion and survival of electrogenic cells 113

of poly-D-lysine (PDL, Sigma, 10 μg/ml in HBSS) and stored in HBSS overnight. The
primary rat embryonic cortical neurons were isolated from CD rat embryos (Charles
River) at the 18th day of gestation (E18) as previously described (Vogt et al., 2003).
Briefly, cells were mechanically dissociated from the isolated cortex tissue by
mechanically triturating in divalent cation free supplemented HBSS [HBSS – Ca2+, Mg2+
(Invitrogen) + 10 mM HEPES (Sigma) + 20 mM Glucose (Sigma) + 1 mM Sodium
Pyruvate (Sigma) + 1 mM Sodium Bicarbonate (Sigma), pH 7.3]. After settling for three
minutes in supplemented HBSS with divalent cations (same formulation as previous
buffer but with Ca2+ and Mg2+), the cells remaining in the supernatant were collected and
counted using trypan blue for viability control. Cells were seeded at approximately
80 cells/mm2 in neurobasal medium containing 2% B27 (Invitrogen), 0.5 mM
L-glutamine (Sigma), and 10 μg/ml gentamycin (Sigma). The neuronal cultures were also
maintained at 37°C in 5% CO2. Medium was exchanged four hours after initial seeding
and then again at day four of culture.

2.3 Fluorescence microscopy

For live-dead staining analysis, 1 mM calcein (Sigma) and 2 mM ethidium

homodimer-1 (EthD-1, Invitrogen) were diluted in 1× Dulbecco’s Phosphate Buffered
Saline solution (DPBS, Invitrogen, Karlsruhe, Germany) + Ca2+, Mg2+ and imaged 10 to
15 minutes after the staining solution was applied. For HEK and HL-1 cells
live-dead staining was performed at three days in vitro (DIV 3). The cell viability of
cortical rat neurons was analysed for three cell cultures on DIV 4 and for two cultures on
DIV 7. For actin staining of neuronal growth cones, cells were fixed with 3.7%
formaldehyde (Sigma), permeabilised with 0.1% triton X-100 (Sigma) + 5% fetal
bovine serum (Invitrogen) in DPBS + Ca2+, Mg2+, and then incubated in
tetramethyl-rhodamine labelled phalloidin from Molecular Probes (Invitrogen) at 20
μg/ml and DAPI (Sigma) at 1 μg/ml. Fluorescence microscopy was performed using an
ApoTome Microscope from Carl Zeiss GmbH (Göttingen, Germany). The images were
analysed with the free software ImageJ (NIH, Maryland) by manually counting the
number of live and dead cells of ten images per sample. Subsequently, analysis of
variance (ANOVA) was performed for 20 pillar and ten planar samples to test for effects
of substrate type, SAM coating and culturing time using SPSS Statistics 19 (IBM
Corporation, USA).

2.4 SEM and FIB imaging of fixed cells

Glutaraldehyde from Agar Scientific Ltd. (Essex, UK) was dissolved in 20 mM HEPES
(pH 7.4 set using 1 mM NaOH). For fixing cortical rat neurons, cells were initially
pre-fixed in 1.25% glutaraldehyde for two hours followed by a fixation in 2.5%
glutaraldehyde overnight. 50% of the glutaraldehyde solution was exchanged with 100%
ethanol for two hours, and then the samples were stored in 75% ethanolic solution
overnight as the result of another serial exchange of the solution. On the following day,
half of this solution was exchanged with 100% ethanol two times, and the samples were
subsequently transferred for critical point drying (CPD 030 from Bal-Tec AG,
Liechtenstein). The ethanol was exchanged against CO2 over one hour, and the samples
114 D. Brüggemann et al.

were dried at the critical point (31°C, 73.8 Bar). Afterwards, the samples were coated
with approximately 20 nm of platinum via sputtering in a SCD 004 system (OC Oerlikon
Balzers AG, Liechtenstein). The HL-1 and HEK cell lines were fixed directly with 3.5%
glutaraldehyde for 4 hours. Subsequently, the same ethanol serial exchange series was
performed as described above. The morphology of the fixed cells on gold nanopillar
arrays and on planar gold was then investigated via SEM. After the SEM analysis,
cross-sections from all cell types on nanopillar arrays were prepared with a FIB. For this
procedure, a second Zeiss Gemini 1550 device was used at the Forschungszentrum
Caesar (Bonn, Germany). The milling of the samples was performed using a focussed Ga
ion beam. Beforehand, the cells were further fixed with Pt from a gas injection system to
avoid their ablation during the milling process.

3 Results

To examine the suitability of gold nanopillars in bioelectronic applications we studied the

adhesion and survival of various electrogenic cells on large-scale gold nanopillar
electrodes. The nanopillars were homogeneously dispersed with a narrow size
distribution. From SEM images (see Figure 1), their average diameter was determined to
be around 60 nm, and the pillars were approximately 300 to 400 nm high with an average
spacing of 25 nm between adjacent pillars (Schröper et al., 2008).

Figure 1 Arrays of gold nanopillars fabricated using a self-assembled nanoporous alumina

template. The pillar diameter is approximately 60 nm and the height is between 300 and
400 nm. (a) top view (b) side view

3.1 Viability of HL-1 and HEK cells on gold nanopillar arrays

For both cell types the cell viability was evaluated at DIV 3 on gold nanopillars and on
planar gold substrates. The fluorescence microscopy study revealed a repeatedly dense
growth of the respective cells on gold nanopillar arrays as well as on planar gold surfaces,
as can be seen for both cell and substrate types in Figure 2. In live cells green
fluorescence was generated by the enzymatic hydrolysis of calcein. Dead cells appeared
red because EthD-1 could enter into the damaged cell membrane, resulting in red
fluorescence from nucleic acid staining. As can be seen, hardly any dead cells were found
in our HL-1 and HEK cultures, neither on gold nanopillars [see Figures 2(a) and 2(c)],
nor on planar gold substrates [see Figures 2(b) and 2(d)].
 Adhesion and survival of electrogenic cells 115

Figure 2 Live-dead staining of HL-1 and HEK cells on gold nanopillar arrays and planar gold
surfaces with EthD-1 and calcein. calcein. (a) HL1-cells on gold nanopillars (b) HEK
cells on nanopillars (c) HL-1 cells on planar gold (d) HEK cells on planar gold (see
online version for colours)

Notes: Live cells appear green and dead cells show red fluorescence. On DIV 3 both cell
lines exhibited a vital and dense growth on the two substrate types.
To study the morphology of HL-1 and HEK cells grown on gold nanopillar arrays, SEM
analysis of the fixed cells was carried out. Thereby, we observed that both HL-1 and
HEK cells grew with protruding filopodiae on gold nanopillars [see Figures 3(a) and
3(c)]. The cell bodies and filopodiae of both cell lines were in close contact with the
nanopillars and showed strong adhesion to the underlying nanostructures. Furthermore,
the cell morphology of HL-1 and HEK cells grown on planar gold surfaces showed the
same result for cell size and shape as on gold nanopillars [see Figures 3(b) and 3(d)].
In the following cross-sectional FIB analysis of the substrates we observed that the
cell membrane of HL-1 cells aligned very well with the underlying gold nanopillar arrays
[see Figure 3(e)]. As Figure 3(f) shows, the HL-1 cell membrane even reached into the
interpillar-spaces with only very small clefts below 100 nm towards the pillar tips. This
indicates very strong adhesion between HL-1 cells and gold nanopillars. FIB
cross-sections of HEK cells on gold nanopillar arrays also yielded a strong coupling
between the cells and the gold nanopillars as can be seen in Figure 3(g). Likewise, the
membrane partly reached into several interpillar regions and clefts below 200 nm
[see Figure 3(h)]. Due to the small distances between cell membrane and nanopillars the
membranes of the two cell lines almost sank into the nanopillar arrays. In several
close-up cross-sections of HL-1 and HEK cells on gold nanopillars we observed shaded
areas directly at the cell membrane, which might indicate a physical penetration of the
nanopillars into the cellular interior. For both HL-1 and HEK cells on planar gold
substrates we observed even tighter coupling without any visible gap between the
substrates and the cell membrane (results not shown).
116 D. Brüggemann et al.

Figure 3 Upper part: SEM images of fixed cells. (a) HL-1 cell on gold nanopillars (b) HL-1 cell
on planar gold (c) HEK cell on gold pillars (d) HEK cell on planar gold. Lower part:
FIB cross-sections through HL-1 and HEK cells on nanopillars show strong adhesion of
the cell lines with very small clefts (below 100 nm for HL-1 cells and below 200 nm for
HEK cells) between the cell membrane and the gold nanostructures, (e) and (f) HL-1
cell on gold nanopillars (g) and (h) HEK cell membrane on gold nanopillars

3.2 Growth of rat cortical neurons on SAM-coated gold nanopillars

The fluorescence microscopy study on the viability of primary rat cortical neurons
revealed strong differences for the underlying substrate. After DIV 4 the survival rate of
neurons on gold nanopillars varied considerably for different experimental runs.
However, on planar gold substrates, the neuronal cell growth could be repeated several
times with similar survival rates. Exemplarily, fluorescence microscopy images of RCN
on gold nanopillars and planar gold coated with NH2 at DIV 4 are shown in Figures 4(a)
and 4(b), respectively. Here, live and dead neurons can be seen at the same time. On
account of the strong variations in the neuronal cell vitality, which we observed, the
cultivation of neuronal cells was extended to DIV 7. For this culture time we found a
 Adhesion and survival of electrogenic cells 117

clear trend of vital neurons on planar gold with pronounced neurite formation in the
reconstructed neuronal network [see Figure 4(d)]. Contrarily, no vital neurons were
observed for gold nanopillars on DIV 7 for most samples [see Figure 4(c)].
Figure 4 The cell viability of rat cortical neurons on gold nanopillars and on planar gold for
DIV 4 and DIV 7. Both substrates in the fluorescence microscopy images (a–d) were
coated with NH2. EthD-1 and calcein were used for live-dead staining, (a) RCN on gold
nanopillars on DIV 4 (b) neuronal cell growth on planar gold on DIV 4 (c) RCN on
gold nanopillars on DIV 7: only dead cells can be seen here (d) dense neuronal cell
growth on planar gold on DIV 7 (e) statistical evaluation of live-dead staining of RCN
on nanopillar arrays (solid bars) and planar gold (dashed bars) in dependence of the
culture time and the respective SAM coating (see online version for colours)

Notes: The cell vitality strongly declined with increasing culture time for neurons grown
on gold nanopillars. However, on planar gold substrates the growth of vital
neuronal cells did not depend on the culture time. This observation was
independent of the two different SAM coatings.
118 D. Brüggemann et al.

For both substrate types and the different SAM coatings the survival of RCN was
analysed statistically for three different cell cultures on DIV 4 and for two cultures
on DIV 7 [see Figure 4(e)]. Independent of the SAM coating the neuronal vitality
was found to be much higher on planar gold (dashed bars) than on nanopillars (solid
bars) for DIV 4 and DIV 7. After three days in culture 54% of the neurons survived
on planar gold with NH2 coating whereas for gold nanopillars with NH2 coating
only 30.4% of the neurons were vital. MML coated substrates yielded similar results
on DIV 4 with 51.1% vital cells on planar gold and only 22% live neurons on
gold nanopillars. The difference in RCN vitality was even more pronounced on DIV 7.
At this cell culture time 38.7% of the neurons survived on planar gold with NH2
coating and only 7.4% vital neurons were found on NH2-coated nanopillars. For
MML coating we found 57.7% vital neurons on planar gold, but only 5.4% of the
neurons on nanopillars were vital. The subsequent ANOVA analysis also revealed that
the factors surface type and culturing time had a significant effect on the neuronal
survival rate (both p ≤ 0.01). Moreover, it was confirmed that the coating did not have
any significant effect on the cell survival (p > 0.05). Thus, gold nanopillar arrays with the
presented geometry did not support vital growth of rat neurons for more than four days in
culture.
Subsequently, nanopillar and planar gold samples, on which vital neuronal cell
growth on DIV 4 was observed, were analysed with regard to the cell morphology using
SEM. A representative SEM image of a rat neuron on gold nanopillars coated with NH2
can be seen in Figure 5(a). Size and shape of the cell body and the neurite outgrowth are
comparable to a neuron grown on a planar gold reference [see Figure 5(b)]. However, we
repeatedly noticed that neuronal cell bodies of RCN grown on nanopillars until DIV 4
had partially detached from the nanostructures. This indicates that these neurons were not
vital any more at the time of fixation, which is in agreement with our previous results
from live-dead staining. Figure 5(c) shows an example of a neuron grown at the border of
gold nanopillars to planar gold. In the planar substrate areas (dark grey) the cell
membrane is connected to the underlying surface very closely by well-spread neurites.
However, when the cell body reaches the horizontal nanostructured region (white dots) it
levitates and the neurite spans the nanostructures until it adheres again in the opposite
planar gold area. Furthermore, hardly any neurite formation can be seen in the
nanostructured part of this sample, indicating less adhesion on the nanopillars than on the
planar gold area.
For further investigation of the neuron-nanopillar interface at DIV 4 FIB
cross-sections were prepared in the regions of interest. Figure 5(d) shows a typical
cross-section of a neuronal cell body on gold nanopillars. A big cleft of 300 to 500 nm
between cell membrane and pillar tips can be seen. In comparison, the FIB cross-section
of a neuronal cell body on planar gold showed no apparent cleft between the cell
membrane and the gold surface, thus indicating strong adhesion [see Figure 5(e)].
Interestingly, cross-sections through neurites on gold nanopillars revealed that the
neurites adhered very closely to the nanostructures and even protruded into the cavities
between vertical nanopillars [see Figure 5(f)]. Thus, our cross-sectional analysis of the
neuron-substrate interfaces confirmed the previously made assumptions and are
consistent with the results from live-dead staining and SEM analysis.
 Adhesion and survival of electrogenic cells 119

Figure 5 SEM images and FIB crosssections of fixed RCN on gold nanopillars and planar gold
grown until DIV 4. (a) RCN on gold nanopillars (b) RCN on planar gold: on both the
planar and the nanostructured gold a comparable neuronal morphology can be seen
(c) RCN grown at the boundary between planar gold (dark grey) and gold nanopillars
(white dots): the cell body adhered tightly to the planar part of the substrate with
neuronal outgrowth whereas the axon bridges the nanostructures and only adheres to the
lower planar gold area (d) cross-section of a neuronal cell body on gold nanopillars: the
cleft of several hundred nanometers between the pillars and the cell membrane indicates
weak adhesion (e) neuronal cell body on planar gold without any cleft showing very
tight adhesion (f) tightly adhered neurite on gold nanopillars partly protruding into the
gaps between the pillars

To investigate the differing adhesion of neuronal cell bodies and neurites on gold
nanopillars in more detail, staining of the actin filaments was carried out. Again, neurons
on gold nanopillars were examined for both SAM types, and the results were compared
with neuronal cell growth on planar gold surfaces. As Figure 6(a) shows exemplarily for
a neuron on nanopillars with MML coating on DIV 4, no growth cone formed at the end
of the neurites. This observation was also made for RCN grown on nanopillar arrays with
NH2 coating. Contrarily to these results, all our fluorescence measurements of RCN on
planar gold showed the formation of widespread growth cones independently of the SAM
coating [see Figure 6(b)]. This observation indicates that changes in the cytoskeleton of
the neurons are induced by the underlying gold nanopillar arrays. These changes in the
actin filaments seem to influence the adhesive response of rat cortical neurons to gold
nanopillars.
120 D. Brüggemann et al.

Figure 6 Staining of neuronal actin filaments for RCN grown on gold nanopillars and planar gold
with MML coating on DIV 4. (a) on gold nanopillar arrays no growth cones were found
and (b) on planar gold substrates the formation of several growth cones was observed
(see online version for colours)

4 Discussion

Several metallic surface modifications have already been examined for cell coupling.
Micron-sized gold protrusions improved the adherence of neurons (Hai et al., 2009a,
2009b; Panaitov et al., 2011), and platinum tip-shaped microelectrodes yielded good cell
coupling in many studies (Heuschkel et al., 2002; Isik et al., 2005; Thiébaud et al., 1997).
Also, with gold nanoflakes dense neuronal networks were obtained (Kim et al., 2010).
However, to date no vertical gold nanostructures have been investigated with regard to
their cell coupling properties, in particular using electrogenic cell types for later signal
recording. Following up our earlier work on microelectrodes modified with gold
nanopillars (Brüggemann et al., 2011) we here present a first study on the cell-adhesive
response of different electrogenic cells to vertical arrays of gold nanopillars.
Our results on the cellular interaction with gold nanopillar arrays show that distinct
cell types react to the vertical gold nanostructures in different ways. This observation is
well in accordance with previous studies using various nanostructured substrate materials
(Lim and Donahue, 2007; Martinez et al., 2009). In our experiments the cell lines HL-1
and HEK 293 showed excellent adhesion and viability on gold nanopillar arrays. In good
agreement with our results for HEK cells are earlier works on HEK cell growth on
SiNWs, which even penetrated into the cell membrane (Kim et al., 2007), and on
nanoporous aluminum oxide (Wolfrum et al., 2006). Two recent studies presented the
reduction of smooth muscle cell proliferation and changes in cell shape when grown on
polymer nanopatterns (Yim et al., 2005) and on TiO2 nanotubes (Peng et al., 2009).
However, this trend was not confirmed in our work with heart muscle cells since we
observed the same growth behaviour and cell morphology for both cell lines on planar
 Adhesion and survival of electrogenic cells 121

and nanostructured gold substrates. In the cross-sections of both cell lines we also
observed very good alignment of the membrane with the underlying nanotopography.
HL-1 cells even protruded into the 20 to 30 nm wide cavities between the pillars, similar
to the interlocked growth of osteoblasts into the pores between TiO2 nanotubes (Oh et al.,
2006). As a result, the HL-1 cells engulfed the nanopillars closely, as it was already
reported for neurons grown on micron-sized gold spines (Hai et al., 2009a, 2009b).
However, to date HL-1 cell membranes had only been found to grow into cavities of
1 μm in diameter (Hofmann et al., 2011). Thus, we could show for the first time that
HL-1 cells aligned into much smaller cavities with dimensions below 100 nm. For future
biosensors, this observation might lead to the fabrication of nanocavity-electrodes with
improved sealing properties, thus enabling improved signal recording and further
electrode miniaturisation.
At many edges of the HL-1 and HEK cell bodies we observed a bending of the
underlying nanostructures in our cross-sections. We assume that this bending originates
from the cell adhesion forces. In future studies the strength of cell adhesion could be
quantified using centrifugal force measurements or staining the focal adhesion points of
these cell lines. Similar results for the bending of underlying nanostructures were
reported previously with vertically aligned carbon and Si nanowires (Li et al., 2009;
Nguyen-Vu et al., 2007). These nanowires have already been used to measure single cell
traction forces. Thus, gold nanopillar arrays could also be implemented into biosensors
for measuring single cell forces in the near future.
From the observation that rat cortical neurons could survive on gold nanostructures
during the first four days of cell culture we conclude that initial adherence to the
nanopillars took place. However, on DIV 4 no growth cones had developed on gold
nanopillars as the actin staining revealed. This observation coincides with an earlier study
on neuronal cell growth on Si nanopillars where axon lengths, neuron polarity as well as
the structure of the neuritic growth cones were changed due to the nanostructures
(Dowell-Mesfin et al., 2004). Furthermore, our observation of neurites protruding into the
pillar arrays coincides well with previous surveys on axon guidance where axons were
shown to internalise single GaP nanowires from a row of nanostructures (Prinz et al.,
2008). Axonal contact guidance could also be achieved with nanoimprinted polymer
patterns (Johansson et al., 2006). To implement these results into future applications with
gold nanopillars, patterns of pillars, as we already presented them (Weber et al., 2011),
might also be used for neuronal growth guiding. This guiding effect could also be
combined with the micropatterning of adhesion promoting proteins (Vogt et al., 2003) or
the nanopatterning of integrin-specific peptides, which was reported to limit the cell
spreading dependening on the pattern dimensions (Arnold et al., 2004; Huang et al.,
2009).
For fibroblasts grown on ZnO nanorods a lack of focal adhesion was reported earlier,
resulting in decreased cell spreading on the nanostructures (Lee et al., 2008). We
therefore assume that the observed lack of focal adhesion points on gold nanopillars at
DIV 4 is responsible for the reduced neuronal cell spreading and the overall death of
RCN on DIV 7. For deposited films of CNTs controversial results on neuronal cell
growth have been reported to date. While good adhesion, neurite outgrowth and the
formation of neuronal networks were observed in several studies (Cellot et al., 2009;
Gabay et al., 2007; Lovat et al., 2005; Mazzatenta et al., 2007) Liopo et al. reported on
reduced neuronal cell attachment and proliferation on single wall CNT films (Liopo
122 D. Brüggemann et al.

et al., 2006). All these studies regarding neuronal growth on CNTs have in common that
the nanostructures were not grown vertically but deposited as films, which might have led
to better neuronal adhesion in many cases. When neurons were grown on vertical arrays
of CNTs they showed good adhesion, and the nanofibres partly impaled the cell bodies
(Nguyen-Vu et al., 2006; Yu et al., 2007). This effect was also observed for different cell
types on vertical SiNWs (Shalek et al., 2010) and on carbon nanofibres (McKnight et al.,
2003). With our cell line experiments using HL-1 and HEK cells we could also see
very close contact between the gold nanopillars and the cell membrane. Many FIB
cross-sections showed that the cell membrane was bending into the interpillar-spaces
letting shaded areas at the pillar tips appear. These areas might be an indicator for partial
incorporation of outstanding pillar tips into the cell membrane as reported by the
previously cited works. Nevertheless, in our study we did not find any neurons being
penetrated by gold nanopillars due to the overall low neuronal adhesion and viability on
our nanostructured substrates. For the investigated cell lines, directed invasive coupling
with the nanostructures might be facilitated by fabricating gold nanopillars with higher
aspect ratios, thus enabling intracellular signal recording. This issue is currently
being addressed in our research work.
Summarising, the various surveys for neuronal growth on different nanostructured
materials strongly indicate that the geometrical and topographical features of the
nanostructures play a significant role in neuronal adhesion and survival as it was also
suggested in Qi’s work with different cell types on SiNW arrays (Qi et al., 2009).
Therefore, in future studies with gold nanopillar arrays a change in pillar geometry might
lead to the formation of neuronal growth cones, which could enhance neuronal adhesion,
cell spreading and viability. The deformation of the neuronal cell membrane on gold
nanopillar arrays could also be investigated with account to the bending stiffness of the
growth cones (Hochmuth et al., 1996). As presented earlier the substrate topography also
plays a critical role in the cell-substrate interaction and the control of different cellular
functions (Curtis and Wilkinson, 1997, 1998; Curtis et al., 2001; Dowell-Mesfin et al.,
2004; Lim and Donahue, 2007; Teixeira et al., 2006). It was found that micropatterns and
anisotropic topography have more an impact on the cell shape whereas nanopatterns and
isotropic topography direct collective cell functions (Lim and Donahue, 2007). Therefore,
a modification of the pillar topography, for instance by nanoshaving, might also yield
different reactions of the neuronal cells. Distinct cells seem to have various chemical
preferences for optimal cell function (Brammer et al., 2011). In previous studies adhesion
promoting proteins such as laminin, fibronectin, poly-lysine or extracellular matrix
proteins have already been used to modify the neuron-electrode distance (Greve et al.,
2007) and to yield faster synapse development, resulting in increased neuronal activity
(Chang et al., 2006; Greve et al., 2007). Thus, these proteins or other SAM coatings
might benefit the neuronal cell response to our large-scale gold nanopillar electrodes as
well.
Another hypothesis for the low viability of rat cortical neurons on gold nanopillars
was that chemical residues from the foregoing pillar production were still present in the
interpillar spaces despite the thorough cleaning step. These chemical remains, i.e., oxalic
and phosphoric acid, cyanide and KOH, might have diffused into the cell layer and
caused neuronal cell death after several days in culture. However, when performing
live-dead staining of the neuronal cell cultures on gold nanopillars at DIV 7 we also
studied the planar gold edges of each chip. In contrast to the area-wide cell death on the
inside gold nanopillar arrays we found vital neurons with neurite outgrowth on the planar
 Adhesion and survival of electrogenic cells 123

gold edges of most chips. This observation shows that the low cell viability and adhesion
of neurons on gold nanopillars is not related to any chemical influences based on
diffusion but most probably originates from the geometrical and topographical
dimensions of the gold nanopillars used in this study.

5 Conclusions

In summary, we have studied the adhesive response of different electrogenic cells to

vertical gold nanopillar arrays for the first time. We could show that large-scale gold
nanopillar electrodes influence the cellular response depending on the cell type of interest
and the respective cell region. For cell lines such as HL-1 and HEK 293 we observed
tight coupling and good alignment at the cell membrane – nanostructure interface. For
HL-1 cells we could even show that their cell membrane protrudes into the nanocavities
between adjacent pillars. This cellular response could be exploited in future extracellular
signal recording and other biosensing applications by enabling high resolution
measurements of cell signals. However, gold nanopillar arrays with the presented
geometries are not yet suitable for coupling to neuronal cells and networks. To enhance
the neuronal cell viability and adhesion on our gold nanostructures, further geometrical,
topographical or chemical modifications of the gold nanopillar arrays will be necessary.

Acknowledgements

The authors acknowledge H.-P. Wingens for the deposition of metal layers and
Angelika Sehrbrock from the Forschungszentrum Caesar, Bonn for preparing the FIB
cross-sections. Financial support was given by the Studienstiftung des deutschen Volkes.

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