Article pubs.acs.org/ac
Electrochemical Imaging of Dopamine Release from ThreeDimensional-Cultured PC12 Cells Using Large-Scale IntegrationBased Amperometric Sensors Hiroya Abe,† Kosuke Ino,*,† Chen-Zhong Li,‡,§ Yusuke Kanno,† Kumi Y. Inoue,† Atsushi Suda,∥ Ryota Kunikata,∥ Masahki Matsudaira,⊥ Yasufumi Takahashi,†,§,# Hitoshi Shiku,† and Tomokazu Matsue*,†,§ †
Graduate School of Environmental Studies, Tohoku University, 6-6-11-604 Aramaki-aza Aoba, Aoba-ku, Sendai 980-8579, Japan Nanobioengineering/Nanobioelectronics Laboratory, Department of Biomedical Engineering, Florida International University, 10555 West Flagler Street, Miami, Florida 33174, United States § WPI-Advanced Institute for Materials Research, Tohoku University, 2-1-1 Katahira, Aoba, Sendai 980-8577, Japan ∥ Japan Aviation Electronics Industry, Ltd. 1-1, Musashino 3-chome, Akishima-shi, Tokyo 196-8555, Japan ⊥ Micro System Integration Center, Tohoku University, 519-1176 Aramaki-aza Aoba, Aoba-ku, Sendai 980-0845, Japan # PRESTO, JST, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan ‡
ABSTRACT: In the present study, we used a large-scale integration (LSI)-based amperometric sensor array system, designated Bio-LSI, to image dopamine release from threedimensional (3D)-cultured PC12 cells (PC12 spheroids). The Bio-LSI device consists of 400 sensor electrodes with a pitch of 250 μm for rapid electrochemical imaging of large areas. PC12 spheroids were stimulated with K+ to release dopamine. Poststimulation dopamine release from the PC12 spheroids was electrochemically imaged using the Bio-LSI device. Bio-LSI clearly showed the effects of the dopaminergic drugs L-3,4dihydroxyphenylalanine (L-DOPA) and reserpine on K+-stimulated dopamine release from PC12 spheroids. Our results demonstrate that dopamine release from PC12 spheroids can be monitored using the device, suggesting that the Bio-LSI is a promising tool for use in evaluating 3D-cultured dopaminergic cells and the effects of dopaminergic drugs. To the best of our knowledge, this report is the first to describe electrochemical imaging of dopamine release by PC12 spheroids using LSI-based amperometric sensors.
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control of cell−cell contact, cell density, and so forth. Baldwin and Saltzman6 investigated dopamine release from 3D-cultured PC12 cells using high-performance liquid chromatography (HPLC) and found that on a per-cell basis, aggregated PC12 cells secrete higher levels of dopamine than do single cells. Their results suggest that the culture method affects dopamine release from dopaminergic cells. Several methods have been developed to evaluate dopamine release. Because dopamine is a redox-active neurotransmitter, electrochemical methods can be complemented with direct electrochemical detection. Electrochemical methods using micro- and nanoelectrodes7−12 offer the advantages of higher spatial resolution compared to methods such as UV and HPLC. In addition, electrochemical methods are suitable for turbid and colored solutions as well as cell suspensions. Using these probe techniques, single exocytotic events can be monitored, which has enabled the elucidation of exocytotic mechanisms.
opamine is a catecholamine neurotransmitter that functions as a chemical messenger in synaptic communication between cells. A shortage of dopamine causes Parkinson’s disease with consequent movement problems.1 Therefore, much attention has been focused on a cell-based therapy for Parkinson’s disease using dopaminergic cells.2−4 To this end, methods have been developed for screening and examining the function of dopaminergic cells. In vitro assays have been developed for investigations of dopamine, and dopaminergic cells, such as in vivo-derived neuron cells, cell lines, and stem cell-derived neurons have been studied using neurospheres.5 PC12 cells (rat pheochromocytoma cells) are often used as a model. In a general assay of dopamine release, PC12 cells are stimulated by injecting a solution of highly concentrated K+; K+ stimulation depolarizes the cells and induces the release of dopamine by exocytosis. Some reports have described dopamine release from dopaminergic cells at the single-cell level. Dopamine release has also been investigated in three-dimensional (3D)-cultured dopaminergic cells. Threedimensional culture is a useful method for enhancing cell function and inducing cell differentiation because it allows for © XXXX American Chemical Society
Received: April 7, 2015 Accepted: May 14, 2015
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DOI: 10.1021/acs.analchem.5b01307 Anal. Chem. XXXX, XXX, XXX−XXX
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Analytical Chemistry
Figure 1. General illustration of the Bio-LSI device and outline of electrochemical imaging of dopamine release from PC12 spheroids. (A) Photographs and micrograph of the device. Red, white, and blue arrows indicate the polycarbonate frame, ceramic substrate, and sensor electrodes, respectively. (B−C) Scheme of cell stimulation. PC12 spheroids were stimulated using method 1 (B) and method 2 (C). (B) Dopamine release was induced by injecting high-K+ buffer in the immediate presence of PC12 spheroids. (C) Dopamine release was induced by introducing PC12 spheroids into high-K+ buffer on the device. (D) Scheme of electrochemical detection of dopamine release from PC12 spheroids. Dopamine was oxidized at 0.60 V vs Ag/AgCl.
mm2), we evaluated the differentiation of 3D-cultured embryonic stem (ES) cells via monitoring of alkaline phosphatase activity.27,28 In this study, we employed the Bio-LSI to detect dopamine release from 3D-cultured cells using PC12 spheroids as a model. PC12 spheroids were stimulated with medium containing a high concentration of K+. Dopamine released by the spheroids in response to K+ stimulation was then oxidized on the sensor electrodes, producing an electrochemical image. We also investigated the effects of the dopaminergic drugs L3,4-dihydroxyphenylalanine (L-DOPA) and reserpine on dopamine release by PC12 spheroids. L-DOPA is a dopamine precursor that is used as a drug for treating Parkinson’s disease. Reserpine inhibits vesicular monoamine transport by displacing catecholamine from neurotransmitter vesicles.11,17
Additionally, probe-based techniques enable scanning for topographical images.8 However, the use of probe electrodes is limited to investigations involving only a few cells due to the labor-intensive nature of having to manually handle each probe. To address this challenge, a variety of systems employing chipbased electrodes for amperometric detection of neurotransmitter release have been implemented.13−17 Individually addressable microelectrode arrays offer many advantages for researchers hoping to achieve rapid, real-time, high-throughput analysis of neurotransmitter release. In addition, by using different microelectrodes in the array, these chip devices are capable of sensing multiple analytes. These devices have been employed to investigate the effect of various dopaminergic drugs on dopamine release from PC12 cells.16 However, addressable microelectrode arrays might not be suitable for screening cells, examining large 3D-cultured cells, or for mapping neurotransmitter release in a cell network because it is difficult to incorporate a large number of sensors into the device due to the lack of sufficient space for lead connectors and connector pads for external devices. For more sensors to be incorporated into a chip device, semiconductor technology has been applied.18−25 Lindau and colleagues recently reported a complementary metal-oxide semiconductor (CMOS)-based chip with 10 × 10 microelectrodes for the detection of dopamine.25 These chip devices are useful for the evaluation of dopaminergic cells. We previously developed a large-scale integration (LSI)-based amperometric device containing 400 sensors with a pitch of 250 μm.26−31 Each sensor in this device, designated Bio-LSI, contains an operational amplifier with a switched-capacitor type I−V converter for in-pixel signal amplification.26 The Bio-LSI has been used for real-time imaging of enzyme activity26,29 and array-based cell analysis.27,28 Because the Bio-LSI device is capable of rapid electrochemical imaging of large substrates (25
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MATERIALS AND METHODS
Bio-LSI Fabrication. Detailed information on the fabrication process is described elsewhere.26,27 Briefly, Pt electrodes, used as sensors, were prepared on Al pads via photolithography and sputtering (Figure 1). SU-8 (SU-8 3005, MicroChem, USA) microwells (diameter, 18 μm; depth, ∼5 μm) were fabricated on the Pt electrodes. The Bio-LSI chips were then affixed to connector pads on a ceramic substrate (Japan Aviation Electronics Industry, Ltd., Japan) (Figure 1A), and a polycarbonate frame (Japan Aviation Electronics Industry, Ltd., Japan) was bonded to the substrate to retain the sample solution at the sensor points (Figure 1A). Electrochemical Detection of Dopamine and Calibration Curve Preparation. Performance of the Bio-LSI was evaluated by monitoring the electrochemical signals associated with dopamine oxidation. Briefly, a reference electrode (Ag/ AgCl/sat. KCl) and a Pt counter electrode were inserted into buffer (Hanks’ balanced salt solution, HBSS; catalog no. B
DOI: 10.1021/acs.analchem.5b01307 Anal. Chem. XXXX, XXX, XXX−XXX
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Analytical Chemistry
Figure 2. Dopamine calibration. (A) Amperograms for 0, 5, 10, 50, 70, and 80 μM dopamine. The potential of all electrodes was stepped from 0.00 to 0.60 V vs Ag/AgCl at 0 s. (B) Dependence of the electrochemical signal on dopamine concentration. Plot shows averaged electrochemical signals from 100 electrodes at the center of the device after potential was applied for 180 s. The error bars indicate ± standard deviations of the data taken from the 100 electrodes.
14025−092, Gibco, USA) containing 0−80 μM dopamine (Sigma, USA) that had been placed on the device. Chronoamperometry was performed by stepping the voltage of all sensor electrodes from 0.00 to 0.60 V vs Ag/AgCl to oxidize the dopamine. Electrochemical signals from each electrode were recorded every 200 ms. Current Simulation. To validate the dopamine calibration curve, redox currents were calculated using COMSOL Multiphysics software (ver. 5.0, COMSOL, Inc., USA). Briefly, 3D models containing microwells were constructed. The diameter and depth of the microwells were 18 and 5 μm, respectively, and the bottoms of the microwells were defined as the electrodes. In the simulation, the electrochemical reaction was assumed to be a two-electron process. The dopamine diffusion coefficient was set to 6.0 × 10−10 m2/s.32 The initial concentration of dopamine in the space was set at 10 μM. The concentration of dopamine at the electrode boundary was 0 mM because the electrode potential was set to be sufficiently positive for the electrochemical reaction. The dopamine diffusion flux was zero at insulating surfaces. The dopamine diffusion flux on the electrodes was calculated to evaluate the current associated with dopamine oxidation at the electrodes. Cell Culture. PC12 cells were donated by the Cell Resource Center for Biomedical Research at Tohoku University. PC12 cells were 3D-cultured using the hanging-drop method33 to prepare PC12 spheroids. Briefly, PC12 cells were suspended in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (PS), and 20 μL droplets containing 100−2000 cells were placed on the cover of a culture dish, which was then inverted so that the droplets hung from the dish cover. The cells were then cultured in a humidified incubator at 37 °C with 5% CO2 for 6 days to allow for the formation of PC12 spheroids. To evaluate the effects of L-DOPA and reserpine on dopamine release, PC12 spheroids formed after 6 days of culture were further incubated in a droplet of culture medium containing 100 μM L-DOPA (Sigma) or 1 μM reserpine (Wako Pure Chemical Industries Ltd., Japan) for 1 h, after which the spheroids were washed with buffer and then stimulated with K+ as described below. Stimulation with K+ and Electrochemical Imaging of + K -Stimulated Dopamine Release. Buffer containing KCl at a final concentration of 105 mM was used to stimulate PC12 spheroids. Prior to stimulation, the reference and counter electrodes were inserted into a sample solution on the device. All sensor electrodes were set to 0.60 V vs Ag/AgCl, and
electrochemical images consisting of 400 electrochemical signals were obtained every 200 ms. PC12 spheroids were stimulated during electrochemical imaging using one of two methods. In method 1, PC12 spheroids were introduced into the buffer on the device, and 5 μL of the high-K+ buffer was then injected for 2−3 s using a micropipette (Figure 1B). In method 2, buffer containing PC12 spheroids was introduced into the high-K+ buffer on the device using a micropipette (Figure 1C). In both methods, K+stimulated dopamine release was monitored electrochemically using the Bio-LSI device. During electrochemical imaging, the PC12 spheroids were also observed under a microscope (Stemi 2000, ZEISS) to acquire visual images of the spheroids on the device. These images were then used to calculate the frontal projected areas of the spheroids. Data Analysis. The highest two current values from the electrochemical images were averaged. The current signal before the stimulation was defined as the background and was subtracted from the peak current (expressed in amperograms) to calculate the Δ current, which was then graphed.
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RESULTS AND DISCUSSION Characterization of the Bio-LSI Device. The Bio-LSI device contains 400 electrochemical detection points on the 18 μm diameter Pt disk microelectrodes (Figure 1). Chronoamperometry analysis of dopamine was conducted to characterize the performance of the device. Figure 2A shows amperograms for various concentrations of dopamine (0−80 μM). The current at 180 s increased almost linearly with the dopamine concentration (Figure 2B), and the detection limit was found to be less than 5 μM, indicating that the device can be utilized for quantitative detection of dopamine. After subtraction of the background current, the current for a 10 μM dopamine sample was 23.9 ± 5.5 pA, which was similar to the simulation current (23.4 pA). We also calculated the theoretical value of a single microdisk electrode with a microwell by the equation i=
4πnFCDr 2 4L + πr
where i is current value, n is number of electrons exchanged, F is the Faraday constant, C is the analyte concentration, D is the diffusion coefficient, r is the electrode radius, and L is the depth of the well.17,34 The theoretical value was 24.4 pA, which was similar to the experimental result and the simulation current. The results indicate that the influence of overlapping diffusion was small in this condition. C
DOI: 10.1021/acs.analchem.5b01307 Anal. Chem. XXXX, XXX, XXX−XXX
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Analytical Chemistry
Figure 3. Electrochemical imaging of dopamine release from PC12 spheroids using Method 1. (A) Micrograph of PC12 spheroids (2000 cells/drop, 6 day culture) on the device. (B) Electrochemical images of PC12 spheroids at various times (0, 10, and 20 s) after injection of buffer as a negative control. The buffer was injected at 0 s. (C) Electrochemical images of PC12 spheroids at various times (0, 10, and 20 s) after injection of high-K+ buffer. (D) Amperogram of oxidation of dopamine released from PC12 spheroids. The PC12 spheroids were stimulated three times by injecting the high-K+ buffer at the times indicated by red arrows.
PC12 spheroids individually using a micropipette. Method 2 was developed to overcome this problem. Electrochemical Imaging of K+-Stimulated Dopamine Release from PC12 Spheroids using Method 2. In Method 2, PC12 spheroids are introduced into a high-K+ buffer on the device to stimulate the spheroids semisimultaneously (Figure 1C). The PC12 spheroids went down quickly to the device surface by approaching the micropipette to the device surface and introducing the buffer containing the PC12 spheroids. Electrochemical signal images acquired after stimulation corresponded to the positions of the PC12 spheroids on the device (Figure 4A and B), indicating that this method is suitable for detecting K+-stimulated dopamine release from spheroids. A previous study showed that in assays of dopamine release from single PC12 cells, spikes of dopamine oxidation occur with a latency of
