Download Cell-based sensor chip for neurotoxicity measurements in drinking

POSTER 2016, PRAGUE MAY 24
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Cell-based sensor chip for neurotoxicity measurements in
drinking water
Dennis Flachs1 and Manuel Ciba1
1
biomems lab, Faculty of Engineering, University of Applied Sciences Aschaffenburg, Germany
[email protected], [email protected]
Abstract. Our drinking water contains residues of
pharmaceuticals. A sub-group of these contaminants are
neuro-active substances, the antiepileptic carbamazepine
being one of the most relevant. For assessment of the
neurotoxicity of this drug at a sub-therapeutical level, a cellbased sensor chip platform has been realized and
characterized. For this purpose, a microelectrode array
chips was designed and processed in a clean room and
optimized in terms of low processing costs and good
recording properties. For characterization of the system
neuronal cells were plated on microelectrode array chips
and electrical activity was measured as a function of applied
carbamazepine concentration. We found that the relative
spike rate decreased with increasing drug concentration
resulted in IC50 values of around 36 µM. This value is five
orders of magnitude higher than the maximal dose found in
drinking water.
Keywords
Microelectrode array, Carbamazepine, neurotoxicity,
cell-based biosensor.
1.
Motivation
Every year large amounts of human and veterinary
pharmaceuticals find their way in our drinking water cycle.
With new and improved analytic methods these drugs can be
detected – even in very low concentrations. These new
options increase the awareness of possible effects correlated
to the chronic uptake of drugs within drinking water.
Although, it is likely that residues in the water have no
adverse effects on the human health, there is no regulation
in terms of critical concentration values [1] due to a lack of
data regarding chronic exposure at low doses.
The number of pharmaceutically active substances
found in drinking water is surprisingly high. More than
twenty different active components and their metabolites
have been detected in drinking water [1]. In this work we
focused on neuro-effective drugs as they have hardly been
addressed in the literature yet. Therefore, we searched
literature for measurements of neuro-effective drugs found
in drinking water [1,2]. Typical compounds are
carbamazepine, diazepam, and diclofenac. Obviously, the
measured concentrations depend strongly on country and the
area where samples were taken. The most relevant substance
identified was carbamazepine. Carbamazepine (CBZ) is a
medication used primarily in the treatment of epilepsy and
neuropathic pain and it is well known to reduce
electrophysiological activity of the brain [3]. In Germany,
CBZ has been identified at twenty-six different samples with
a maximum concentration of 0.03 µg/l (0.13 nM) [1]. Our
hypothesis is that CBZ, if incorporated on a regular base –
even at sub-therapeutical levels – has the potential to induce
unwanted changes in the brain’s functionality.
With this background, we propose the application of a
cell-based in vitro assay to assess the neurotoxicity of CBZ
and as a perspective other neurotoxins relevant for drinking
water. For this purpose a cell-based sensing platform shall
be developed. Microelectrode array (MEA) chips coupled to
neuronal cell networks are a common approach to achieve a
basic understanding of electrophysiological properties of
neuronal networks [4,5]. Electrical activity of a neuronal
network is recorded in vitro and changes related to the
application of a neuroactive substance are analyzed. With
this goal, we fabricated microelectrode array (MEA) chips
with gold electrodes to record electrophysiological signals
of a cortical neuronal network and characterized the cellbased sensing platform for the drug CBZ.
2.
Materials and Methods
2.1.
MEA Chip processing
The microelectrodes for recordings of neuronal signals
have been processed of various metals or conduction
polymers [6]. In this study an array of cost-effective
microelectrodes was processed with chrome-nickel-gold
microelectrodes. Fig. 1 shows the layout of the MEA-chip.
The dimensions of the glass chip are 45 mm ∙ 45 mm
providing 59 microelectrodes with a diameter of 30 µm (and
a spacing of 200 µm) and a reference electrode. All
electrodes are connected via conducting lanes to large metal
pads at the outer boarder of the chip, while insulation and
biocompatibility are provided by a thick resist SU-8.
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D. FLACHS, M. CIBA, CELL-BASED SENSOR CHIP FOR NEUROTOXICITY MEASUREMENTS IN DRINKING WATER
container for the cell medium. In Fig. 3 a completely
assembled chip is shown.
Fig. 1. Layout of the microelectrode-array. The array consists of 59
microelectrodes and a larger reference electrode (not shown).
Chips were fabricated in clean room with the process
depicted in Fig. 2. First, a 20 nm thick chrome adhesion
layer was deposited on a four-inch glass wafer by thermal
evaporation using chrome-plated tungsten rods (Kurt J.
Lesker Ltd., Hastings, England). Next, a nickel layer
(80 nm) was also evaporated from 99.995% pure nickel
pellets (Lesker), see Fig. 2a. Now the metal layers were
photo-lithographically structured with the positive
photoresist ma-P 1215 (micro resist technology GmbH,
Berlin, Germany), see Fig. 2b, and exposed to UV light
(365 nm) in a UV Kub (Kloé, Montpellier, France). The
resist was developed in ma-D 331 (micro resist technology)
for 90 seconds. Finally, chrome and nickel layers were
chemically wet etched in a mixture of phosphoric acid
(H3PO4), nitric acid (H3PO4), acetic acid (CHCOOH), and
water (H2O) (3:3:1:1) for nickel, and perchloric acid, ceric
ammonium nitrate, and water (4.25:10.9:84.85) for chrome,
Fig. 2c. After etching, the remaining resist was removed, Fig
2d.
To ensure biocompatibility of the electrodes, they are
covered with a thin film (50 nm) of gold. For that purpose a
currentless, galvanic method was applied, Fig. 2e. The last
step was the deposition of an electrical insulation and
passivation layer. For that purpose, a thick, negative resist
SU-8 (Micro Chem Corp., Westborough, USA) was spin
coated at 3500 U/min resulting in a thickness of 1.5 µm. SU8 is chemically stable and shows excellent dielectric
properties (εr= 3.28 at 1 GHz). Here it was an elegant and
low cost replacement of more common dielectric materials
like CVD silicon nitride, Fig. 2f. Photo-lithographical
structuring of the SU-8 layer allowed to open the
microelectrodes, Fig. 2g, before chips were diced with a
wafer saw (Disco Inc., Santa Clara, USA). A glass ring glued
with polydimethylsiloxane (PDMS) onto the chip, offers a
Fig. 2. Schematic illustration of the MEA-chip fabrication
process. (a) deposition of chrom and nickel on a glass
wafer coated with photoresist, (b) structuring of the
photoresist, (c) etching of chrome and nickel, (d)
removing of photoresist, (e) currentless, galvanic
deposition of a thin gold layer, (f) deposition of the SU-8
passivation layer, (g) structuring of the SU-8 layer.
2.2.
Impedance spectroscopy
Electrochemical impedance spectroscopy was
performed with a VersaSTAT 4 (Princeton Applied
Research) in a scanning range of 0.1 Hz to 1 MHz and signal
amplitudes of 20 mV. Measurements were conducted in
phosphate-buffered saline (PBS) using an Ag/AgCl pellet
counter/reference electrode (Multi Channel Systems,
Reutlingen, Germany) in a two-electrode set-up.
2.3
Neuronal Cell Culture
Neural cell culturing was performed by using cryoconserved embryonic cortical rat neurons (E18 and E19)
(Lonza Ltd, Basel, Switzerland). Cell culture medium was
prepared according to the following protocol: L-glutamine
(2 mM) and 2% NSF-1 were added to the PNBM basal
medium. NSF-1, a supplement supporting neuronal growth
and survival, was aliquoted, frozen and added to the medium
immediately before each use. Neurons were thawed and
cultured with a density of approximately 5.000 cells/mm2
onto each substrate and incubated in a humidified
atmosphere for 4 h. Finally, petri dishes were filled with
approximately 3 ml culture medium. Half the medium was
POSTER 2016, PRAGUE MAY 24
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replaced twice a week and completely replaced every second
week.
2.4
MEA recording
Neuronal signals were recorded extracellularly at a
sampling rate of 10 kHz with a MEA60 amplifier system
(Multichannel System, Reutlingen, Germany) and stored for
offline analysis with the custom-made software tool DrCell
[6] covering signal detection as well as spike-rate and burstrate determination. Signal acquisition was conducted
between 21 and 25 days in vitro (div). Recordings were
performed outside the incubator. To maintain the sample
temperature at 37°C, a temperature-controller (TC01,
Multichannel Systems, Reutlingen, Germany) was
employed during the recordings. Controlled CO2
atmosphere (5%) was provided by a custom-made mini
incubator and a CO2 controller (CO2-Controller 2000,
Pecon, Erbach, Germany).
Test substance CBZ (CAS number 298-46-4; C4024-1G,
Sigma Aldrich, St. Louis, USA) was diluted in
dimethylsulfoxide (DMSO) (CAS number 67-68-5;
Sotrachem, Saint Denis, France) and was manually applied
to the medium. CBZ concentration of 2 µM, 20 µM, 40 µM,
60 µM, 100 µM, 200 µM and 1000 µM were added in an
accumulative manner and recorded for 5 minutes each.
Maximum DMSO concentration at 1000 µM CBZ was
0.5%, which has been shown to have no significant effect on
neural activity [3].
2.5
noise ratio. To improve the impedance, the electrodes may
be coated with nanowires or carbon black to increase the
surface area of the electrodes [7].
Statistics
Data (given as mean ± standard deviation of N samples) was collected from different cell cultures. Data points
of dose-response curves for electrophysiological recordings
were fitted by using the Hill equation
𝑦 = 𝐴2 +
𝐴1 −𝐴2
𝑐 𝑛𝐻
𝑐0
1+( )
Fig. 3. Microelectrode array (MEA) chip (left) and magnified
part of the electrode array (right). Electrodes feature a
diameter of 30 µm and are overgrown by cortical neurons
which form a neuronal network (21 days in vitro).
3.2
Drug Response Curve
The acute application of CBZ to neurons coupled to
MEA chips (N = 8, 21 – 25 div) caused a concentration
dependent decrease in signal activity, which was expected
since CBZ blocks sodium channels. Fig. 4 shows the CBZ
concentration dependent spike rates (mean ± standard
deviation) normalized to the control samples (with a
concentration of zero).
(1)
where c donates the concentration of the test substance, c0
the half maximal effective concentration (designated as EC50
for excitation and IC50 for inhibition), nH the Hill coefficient,
and A1 and A2 the spike rate under control conditions and
following a saturating concentration of the test substance.
3.
Results and Discussion
3.1
MEA Chip
An impedance spectroscopy was conducted to examine
the quality of the microelectrodes. Result revealed an
impedance of about 800 kΩ ± 53 kΩ at 1 kHz. This
frequency is a common reference parameter to compare
recording electrodes. Compared with commercially
available MEA chips (e.g. Multichannel Systems), the
impedance is about two times higher. This relatively high
impedance may be disadvantageous in terms of signal-to-
Fig. 4. Effect of increasing concentrations of CBZ on the spike
rate of neurons coupled to MEA chips (N = 8, 21 – 25 div).
Spike rate (mean ± standard deviation) is normalized to
the control at zero concentration (not shown in the
diagram) for every electrode. Data are fitted using the Hill
equation to calculate half maximal inhibitory
concentration (IC50).
Relative spike rates were calculated for every active
electrode and subsequently averaged across the chip. Fitting
with Hill equation resulted in IC50 values of around 36 µM
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D. FLACHS, M. CIBA, CELL-BASED SENSOR CHIP FOR NEUROTOXICITY MEASUREMENTS IN DRINKING WATER
which is in agreement with values reported for primary
murine frontal cortex neurons [4]. Compared with
concentrations measured in drinking water, the IC50 value is
five orders of magnitude higher. Therefore, no acute effects
on neurons are expectable due to exposure of CBZ in
concentrations measured in drinking water. Since neurons
coupled to MEA chips can be cultured for several weeks and
are capable of capturing the effect of active components like
CBZ, they might be also applied to study acute or long-term
effects of certain mixtures found in drinking water.
Summary
physiologically based neurotoxicity testing platform for the 21st
century. Neurotoxicology, 2010, vol. 31, no. 4, p. 331–350.
[5] DAUS A.W., LAYER P.G., THIELEMANN C., A spheroid-based
biosensor for the label-free detection of drug-induced field potential
alterations. Sensors and Actuators B: Chemical, 2012, vol. 165, p. 5358.
[6] NICK, C., GOLDHAMMER M., BESTEL R., STEGER F., DAUS A.
W., THIELEMANN C., DrCell – A Software Tool for the Analysis
of Cell Signals recorded with Extracellular Microelectrodes
Signal Processing: An International Journal, 2013, vol. 7, no. 2, p.
96-109.
[7] NICK C., QUEDNAU S., SARWAR R., SCHLAAK H. F.,
THIELEMANN C., High Aspect Ratio Gold Nanopillars on
Microelectrodes for Neural Interfaces. Microsystems Technologies,
20 (10-11), 1849-1857, 2014, DOI: 10.1007/s00542-013-1958-x.
We realized an in vitro cell-based sensing platform in
order to assess neurotoxicity of active components that are
relevant contaminants of drinking water.
A MEA chip process was developed to process lowcosts sensors for less than 40 € (made in the clean room of
the faculty), avoiding expensive process steps like gold
evaporation and CVD processes. The impedance of the
microelectrodes is only two times higher than commercially
available TiN microelectrodes. This disadvantage may be
easily overcome in future work by electrochemically
deposited nanowires.
Neurons coupled to MEA chips were electrical active
and signals could be recorded and analyzed. The acute
application of the neuroactive substance CBZ caused a
concentration dependent decrease in signal activity (spike
rate) with a IC50 value of around 36 µM, which was expected
[4]. Further experiments will now address the chronic
application of CBZ for several days and weeks.
Acknowledgements
Research described in the paper was supervised by
Prof. C. Thielemann, biomems lab, UAS Aschaffenburg and
financially supported by the Bayrisches Staatsministerium
für Bildung, Kultus, Wissenschaft und Kunst in the frame of
FH Forschungsschwerpunkt. We would like to thank Dr.
CRISTOPH NICK for the making of the MEA chip layout.
References
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About Authors
Dennis FLACHS was born in Aschaffenburg, Germany,
and earned his bachelor degree as industrial engineer in
March 2016 at the UAS Aschaffenburg. Currently he is enrolled in the scientific master program of electrical engineering at the same university.
Manuel CIBA was born in Erlenbach a. M., Germany. He
completed his Master of Engineering in 2014 at the UAS
Aschaffenburg. Currently, he is working on his PhD in
cooperation with the JMU Würzburg.