Printed Carbon Microelectrodes for Electrochemical Detection of

Technical Note pubs.acs.org/ac

Printed Carbon Microelectrodes for Electrochemical Detection of Single Vesicle Release from PC12 Cells Alexey Yakushenko, Jan Schnitker, and Bernhard Wolfrum* Peter Grünberg Institute (PGI-8/ICS-8), Forschungszentrum Jülich, 52425 Jülich, Germany JARA- Fundamentals of Future Information Technology, Forschungszentrum Jülich, 52425 Jülich, Germany ABSTRACT: We present a disposable system for recording neurotransmitter release from individual cells in vitro. A simple yet reliable microelectrode fabrication process is introduced using screen-printed carbon paste. It allows rapid fabrication of devices at low costs without standard clean-room technology. We demonstrate functionality of the system by real-time observation of vesicle release from single PC12 (rat pheochromocytoma) cells. The cells are cultured directly on the chip and can be used for immediate or long-term in vitro experiments. Thus, our approach may serve as a platform for pharmacological cell culture studies.

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passivation deterioration.15,16 According to our experience with MEAs, a typical chip cannot be reused more than approximately 5 times in long-term cell culture experiments of several weeks. For this reason, the use of low-cost disposable systems is desirable for high-throughput cell culture analysis. In this technical note, we present single-vesicle release recordings from a catecholamine-containing PC12 (rat pheochromocytoma) cell line cultured directly on a disposable screen-printed carbon paste microelectrode. The carbon paste microelectrode fabrication process is carried out in a standard chemical laboratory with no special clean-room equipment. Total material cost of a single device with all required components amounts to approximately 20 cents per chip. Additionally, the microelectrode fabrication process including first in vitro dopamine recordings from PC12 cells can be achieved in less than 6 h.

echniques for monitoring vesicular release of neurotransmitters can be used to investigate processes related to chemical signal transduction between neurons or neuron-like cell lines. Diverse electrochemical methods are particularly suited to detect readily oxidizable neurotransmitters such as dopamine, adrenaline, and serotonin.1,2 Single vesicle release of neurotransmitters has been intensively studied using amperometric techniques.3−10 In amperometry, the potential of the working electrode is kept constant against a reference electrode, namely, above or below the redox potential at which the molecule of interest is oxidized or reduced, respectively. The technique has the advantage of high temporal resolution and sensitivity while lacking selectivity toward different molecules with similar redox potentials. The working electrode of choice for such measurements is usually a carbon-fiber microelectrode, which exhibits a wide potential window and inert electrochemistry.11 On the other hand, carbon microelectrodes cannot be easily integrated on a chip for direct cell culturing. Planar metal microelectrodes, which are extensively used for measurements of electrical activity of cells in the neurophysiological community,12 are more prone to chemical fouling after prolonged exposure to electrolytes and have a narrower potential window that limits their application. Nevertheless, metal electrode arrays enable direct cell culture growth and in vitro detection of single vesicle release as demonstrated by Hafez et al.13 Since the discovery of pyrolyzed photoresist film (PPF) microelectrodes by the group of Madou,14 there is a possibility to build arrays of carbon-based electrodes directly onto a planar substrate. However, both types of electrode arrays require optical lithography or similar clean-room facilities for fabrication. This makes the production cost of planar microelectrode arrays (MEA) relatively high. Commercially available 64 channel MEAs are sold for more than $100 per chip. After long exposure times to electrolyte solution, the performance of the chip degrades due to electrode or © 2012 American Chemical Society

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EXPERIMENTAL SECTION Materials. Polydimethylsiloxane (PDMS, Sylgard 184) was obtained from Dow Corning GmbH (Wiesbaden, Germany). A mixture of elastomer and curing agent in a ratio of 10:1 was prepared and stored in a freezer at −20 °C before use. Microscope slides were purchased from Thermo Scientific (Waltham, MA) and used without any further cleaning. Silver glue (Epo-Tek H20E-PFC) was purchased from Epoxy Technology (Billerica, MA). Parts of a biomedical sensor materials demonstrator sheet, kindly provided for testing by DuPont Microcircuit Material (DuPont MCM, Wilmington, DE) were used as the electrode material. The demonstrator sheet included strips of screen-printed and cured carbon Received: February 16, 2012 Accepted: April 18, 2012 Published: April 18, 2012 4613

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Technical Note

(BQ221) and Ag/AgCl (5874) pastes on polyethylene terephthalate (PET, Melinex, DuPont, Wilmington, DE). Dulbecco’s modified Eagle’s medium (DMEM), fetal calf serum (FCS), penicillin-streptomycin (Pen/Strep) mixture, and poly-L-lysine (PLL) were obtained from Sigma (St. Louis, MO) and used without further purification. Fabrication of Planar Microelectrodes. The screenprinted and cured carbon and Ag/AgCl pastes on PET sheets were cut to yield strips of approximately 0.5 mm × 10 mm size. The strips were subsequently glued with PDMS onto a microscope slide in a perpendicular arrangement and cured in the oven for 1 h at 150 °C. Then a glass ring (9 mm diameter) was glued on top of the strips followed by 1 h curing at 150 °C to serve as a reservoir for future cell culture studies. Finally, a thin layer of PDMS (>6 μm) was added inside the ring to cover the carbon strip, whereas the Ag/AgCl electrode inside the ring was left exposed to form a large reference electrode area. Additionally a drop of silver glue was applied to the free ends of carbon and Ag/AgCl strips outside the reservoir to form reliable bond pads. The whole device was again cured for 1 h at 150 °C. Afterward, a probe head (SUSS MicroTec AG, Garching, Germany) equipped with a tungsten needle was used to pinch through the PDMS layer and expose the carbon working electrode surface. The needle was positioned vertically above the electrode using a stereomicroscope and lowered into the PDMS forming a hole. During this process, the resistance across the carbon electrode and the tungsten needle was monitored. Upon approaching the electrode surface, a sharp decrease in resistance was observed and the needle was retracted after the resistance stabilized below 10 kΩ. Figure 1 shows a picture of a finished carbon paste microelectrode chip compared to a MEA fabricated using standard clean-room processes as previously described.17

Table 1. Cost Calculation for a Single Printed Microelectrodea material

quantity used per chip

cost per chip

carbon paste Ag/AgCl paste PDMS silver glue glass slide glass ring total:

10 mg 10 mg 50 mg 20 mg 1 piece 1 piece

$0.006 $0.012 $0.005 $0.061 $0.076 $0.040 $0.20

a

The work time required for a single chip is approximately 30 min for an experienced user, excluding waiting times during curing.

used as the electrolyte in all measurements. To assess the quality of the insulation layer, we performed amperometric recordings with a patch clamp amplifier (EPC-10 Quadro PCI, Heka Elektronik Dr. Schulze GmbH, Lambrecht, Germany) using a 1 mM 1,1′-ferrocenedimethanol (Sigma) in 100 mM KCl solution. The results were compared before and after introducing the microapertures in the PDMS passivation. Cell Culture. In vitro experiments were performed on a rat pheochromocytoma cell line (PC12) capable of releasing neurotransmitters such as dopamine, kindly provided by Agnes Dreier and Joachim Weis at Uniklinikum Aachen, Germany. A stock PC12 culture was grown in Petri dishes using DMEM medium supplemented with 10% FCS and 1% Pen/Strep, kept in the incubator at 37 °C and 5% CO2 and was passaged every 3−4 days. Before cell plating, chips were sterilized by UV light for 30 min. Afterward, the chips were incubated for 30 min with 80 μL of 10 μg/mL solution of PLL to enhance cell adhesion. Cells were trypsinated from an 80% confluent cell culture in a Petri dish and collected into a 15 mL Falcon tube. After centrifugation, the cell pellet was resuspended in 1 mL of supplemented DMEM medium. Subsequently, 50 μL of supplemented DMEM medium were added inside the ring of the chip. A volume of 20 μL of cell suspension was pipetted into the reservoir, and the cells were allowed to sediment and adhere in the incubator at 37 °C for 2 h. Data Acquisition. The HEKA patch clamp amplifier system was also used to record current traces during in vitro experiments. Both the on-chip carbon working electrode and the Ag/AgCl reference electrode were connected to the corresponding slots of the head-stage preamplifier. The working electrode was used in amperometric mode and kept at a constant potential of either 0 or +1 V against the reference electrode. The measurement time resolution was set to 20 μs. The signal was low-pass filtered with two built-in hardware Bessel filters of the patch clamp amplifier first at 10 kHz and then at 0.1 kHz. The data was exported into Matlab (The MathWorks Inc., Natick, MA) for postprocessing.

Figure 1. (Left) Printed microelectrode with carbon working electrode (black) and Ag/AgCl reference electrode (dark gray). (Right) Standard clean-room fabricated 64-channel microelectrode array (MEA). Scalebars: 5 mm.

In addition to simple and fast fabrication, the materials for building microelectrodes by this design are very cheap. Table 1 shows approximate material costs per fabricated device. Values are given according to estimates of average quantities used per chip. Microelectrode Characterization. Scanning electron microscopy (SEM) was performed for characterization of the electrode geometries and aperture sizes. The passivation layer thickness was measured with a stylus profilometer (Dektak 150, Veeco, Plainview, NY). A potentiostat (PGSTAT30, ECO CHEMIE B.V, Utrecht, The Netherlands) was used for electrochemical impedance spectroscopy (EIS) of the electrodes. The measurements were carried out in a three-electrode mode with a platinum counter electrode. HEPES buffer was

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RESULTS AND DISCUSSION SEM Characterization. SEM imaging was performed to characterize the screen-printed carbon paste microelectrodes. Pinching the holes with a probe head under current control resulted in small but regular apertures with an average maximum opening of 2.80 ± 0.76 μm (n = 5). Figure 2 shows an image of a typical microelectrode. A PDMS flap was observed adjacent to the aperture for all chips. This flap was caused by the pinching process and did not pose a problem to actual cell measurements.

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capacitances may be explained by the lower roughness of the BQ221 carbon electrode. The electrodes used by Cheng et al. for example, display a very granuolus surface with spheres of several hundred nanometers in diameter. On the contrary, the carbon paste electrodes used in our experiments exhibit a smoother surface without visible granules as can be seen on the inset of Figure 2. A smooth electrode surface is beneficial for current measurements where the noise is dominated by the interfacial capacitance of the electrode−electrolyte interface.22 We performed amperometric measurements in 100 mM KCl solution with 1 mM 1,1′-ferrocenedimethanol to electrochemically characterize the microelectrodes and PDMS passivation layer. The background-corrected steady-state currents before and after introducing the apertures were 830 zmol). Partially, these events might represent several superimposed released vesicles, which are temporally indistinguishable in the data trace. In this case, the current peaks have a longer duration (>15 ms). Nevertheless, most “giant” peaks have a half-width identical to the duration of the regular peaks, indicating that they belong to a single vesicular release event. Such a high vesicular content is more characteristic to chromaffin cells3 (3.1 million molecules/vesicle or 5.2 amol) than to PC12 cells.26 However, differences in vesicular content of different PC12 subcultures might be responsible for these deviations.27 The possibility of detecting single vesicle release instead of neurotransmitter bulk recordings extends the scope of applications for printed carbon electrodes.28

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Address: Peter Grünberg Institute (PGI-8/ICS-8), Forschungszentrum Jülich, 52425 Jülich, Germany. Phone: +49-2461-61-3285. Fax: +49-246161-8733. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Enno Kätelhön and Andreas Offenhäusser for helpful discussions and Agnes Dreier and Joachim Weis for providing the PC12 cells. This work was funded by the Helmholtz Young Investigators program.

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REFERENCES

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