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POSTER 2016, PRAGUE MAY 24 1 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. 2 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 3 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 4 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 [1] BERGMANN A., FOHRMANN R., WEBER F.A., Zusammenstellung von Monitoringdaten zu Umweltkonzentrationen von Arzneimitteln. Umweltbundesamt, 2011. [2] WEBB S., TERNES T., GIBERT M., OLEJNICZAK K., Indirect human exposure to pharmaceuticals via drinking water. Toxicology letters, 2003, vol. 14, no.3, p.157–167. [3] SHEETS P.L., HEERS C., STOEHR T., CUMMINS T.R., Differential block of sensory neuronal voltage-gated sodium channels by lacosamide [(2R)-2-(acetylamino)-N-benzyl-3methoxypropanamide], lidocaine, and carbamazepine. Journal of Pharmacology and Experimental Therapeutics, 2008, vol. 326, no. 1, p. 89–99. [4] JOHNSTONE A. F., GROSS G. W., WEISS D. G., SCHROEDER O., GRAMOWSKI A. SHAFER T. J., Microelectrode arrays: a 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.