Integrated Microdevice for Long-Term Automated Perfusion Culture

ARTICLE pubs.acs.org/ac

Integrated Microdevice for Long-Term Automated Perfusion Culture without Shear Stress and Real-Time Electrochemical Monitoring of Cells Lin-Mei Li,† Wei Wang,† Shu-Hui Zhang,† Shi-Jing Chen,† Shi-Shang Guo,‡ Olivier Franc-ais,§ Jie-Ke Cheng,† and Wei-Hua Huang*,† †

Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, and ‡College of Physical Sciences and Technology, Wuhan University, Wuhan, 430072, China § SATIE, UMR 8029 CNRS, Ecole Normale Sup
erieure de Cachan, 61 Avenue du Pr
esident Wilson, 94235 Cachan cedex, France

bS Supporting Information ABSTRACT: Electrochemical techniques based on ultramicroelectrodes (UMEs) play a significant role in real-time monitoring of chemical messengers’ release from single cells. Conversely, precise monitoring of cells in vitro strongly depends on the adequate construction of cellular physiological microenvironment. In this paper, we developed a multilayer microdevice which integrated high aspect ratio poly(dimethylsiloxane) (PDMS) microfluidic device for long-term automated perfusion culture of cells without shear stress and an independently addressable microelectrodes array (IAMEA) for electrochemical monitoring of the cultured cells in real time. Novel design using high aspect ratio between circular “moat” and ring-shaped micropillar array surrounding cell culture chamber combined with automated “circular-centre” and “bottom-up” perfusion model successfully provided continuous fresh medium and a stable and uniform microenvironment for cells. Two weeks automated culture of human umbilical endothelial cell line (ECV304) and neuronal differentiation of rat pheochromocytoma (PC12) cells have been realized using this device. Furthermore, the quantal release of dopamine from individual PC12 cells during their culture or propagation process was amperometrically monitored in real time. The multifunctional microdevice developed in this paper integrated cellular microenvironment construction and real-time monitoring of cells during their physiological process, and would possibly provide a versatile platform for cell-based biomedical analysis.

E

lectrochemical detection based on ultramicroelectrodes (UMEs) has become one of the most powerful techniques in real-time monitoring of an infinitely minute number of released molecules from single cells and investigation of the mechanisms of the vesicular exocytosis as well.1
9 Much important quantitative (e.g., maximum flux and total number of chemical messengers released) and kinetic (e.g., fusion pore formation, various modes of exocytotic events, and corresponding time variables) information associated with single exocytotic events has been obtained on various type of cells with high spatial and temporal resolutions.2,5,9 However, as the probably most widely used method for the study of single exocytotic events, the carbon fiber microelectrodes (CFMEs) based electrochemical technique is, to a certain extent, a labor-intensive and low-throughput technique, since the electrodes must be precisely controlled by micromanipulators and only one cell can be detected at a time. In addition, as an important complementary technique for investigation of exocytosis, the fluorescence imaging of the same electrochemically detected vesicles could not be simultaneously achieved. An alternative method for electrochemical detection is the use of the planar microfabricated microelectrode or microelectrode r 2011 American Chemical Society

arrays (MEAs), by which the cells are directly located or trapped on microfabricated electrodes without highly accurate positioning techniques,7,8 which facilitates the electrochemical monitoring to be performed in a facile and high-throughput manner. Since the initial demonstration by Cooper’s group utilizing a microfabricated micrometer-scale chamber containing an integrated electrochemical sensor for enzyme-mediated electrochemical detection of purine release from single rat cardiomyocyte,10 an increasing variety of microfabricated devices for electrochemical detection of cells have been recently reported. Several kinds of materials including gold (Au),11
15 platinum (Pt),16
20 indium
tin oxide (ITO),21
24 nitrogen-doped diamond-like carbon (DLC:N),25
27 boron-doped nanocrystalline diamond (NCD),28 and functionalized carbon nanotubes (CNTs)29 have been deposited on silicon or glass wafers to form size-controllable and shape-controllable electrodes by microfabrication technologies, and well-resolved Received: August 30, 2011 Accepted: November 16, 2011 Published: November 16, 2011 9524

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Analytical Chemistry quantal amperometric events were obtained in a high-throughput manner. The electrochemical monitoring of exocytosis using a microfabricated device so far is mainly performed by placing or trapping single or clusters of cells on electrodes with microfabricated structures. However, cells in vivo are in a complicated “bio-microfluidic” cellular microenvironment.30,31 Precise monitoring of cells in vitro absolutely depends on the successful construction of cellular physiological microenvironment. Recent microfluidic or lab-on-a-chip (LOC) devices provide powerful tools and offer an increasingly versatile series of platforms for cell analysis.32
35 Application of lab-on-a-chip technology combining MEAs for electrochemical detection and microfluidic channels for cell loading/trapping and reagents/secretagogue perfusion offers a compact and automated monitoring technique. Furthermore, by biofunctionization of the surfaces and control of extracellular matrix, microfluidic devices provide the capability of mimicking the natural physiological environment which cells are subjected to. Coupling of MEAs with microfluidics would provide the capability to probe cells during their cellular physiological process (e.g., cell growth, development, and cell signal transduction) in real time. Incorporation of MEAs into a microfluidic platform has been used for investigation of exocytosis, in which the microfluidic channel was mainly used to automatically handle cells and solutions.14,18,19,22,23,36 However, there are very few papers that reported utilizing a highly integrated microdevice composed of microfluidics and MEAs that are capable of realtime monitoring of the cells during their physiological process, and the unique advantage of microfluidics on mimicking the in vivo cellular situations in an integrated MEAs
microfluidics device is vastly underexplored. In this paper, we fabricated a highly integrated multilayer microsystem composed of a poly(dimethylsiloxane) (PDMS) microfluidic device for long-term automated perfusion culture of cells and an independently addressable microelectrodes array (IAMEA) for real-time electrochemical monitoring of cells. This study is aimed at developing a multifunctional microdevice capable of real-time monitoring cells during their cellular physiological process in an automated and high-throughput manner. The versatility of the microdevice was demonstrated by performing long-term automated cultures of human umbilical endothelial cell line (ECV304) and neuronal differentiation of rat pheochromocytoma (PC12) cells. Furthermore, the quantal release of dopamine from the cultured single PC12 cells was amperometrically monitored in real time.

’ EXPERIMENTAL SECTION Materials and Reagents. Dopamine (DA), poly-L-lysine (PLL), laminin, fluorescein isothiocyanate (FITC), and neuron growth factor (NGF) were purchased from Sigma (St. Louis, MO), RPMI 1640 and DMEM/F12 medium for cell culture were purchased from GIBCO, L-glutamine and HEPES were purchased from Amresco, 30 ,60 -di(O-acetyl)-40 ,50 -bis[N,N-bis(carboxymethyl)-aminomethyl] fluorescein, tetraacetoxymethyl ester (calcein-AM), and 3,8-diamino-5-[3-(diethylmethylammonio)propyl]-6-phenylphen-anthridinium diiodide (PI) for cell staining were obtained from Dojindo laboratory, U.S.A. PC12 cells and ECV304 cells were from China Center for Type Culture Collection (CCTCC, China). All other chemicals unless specified were reagent grade and were used without further purification.

ARTICLE

Multilayer Microdevice Construction. The microdevice we fabricated here consists of a borosilicate glass substrate (4  4 cm2) with Ti/Pt MEA on it (Figure 1a) and an SU-8 2002 photoresist insulating layer (Figure 1b), two PDMS layers microfluidic parts with imprinted microfluidic channels and chambers (Figure 1, parts d and e), and a spin-coated thin PDMS layer (ca. 20 μm thick, Figure 1c) to promote the bonding between MEA and the microfluidic chip. Fabrication of Microelectrode Arrays. The 6 6 platinum circular microelectrodes were patterned onto the glass substrate and the conducting lines were insulated using standard photolithography techniques (Supporting Information Figure S1). Masks for making MEAs and insulating units were obtained by printing a specific pattern onto a transparency film with a highresolution (20 000 dpi) printer. A thin layer of positive photoresist AZ 5214E (Clariant, U.S.A.) was spin-coated on a cleaned glass substrate (4  4 cm2, 300 μm thick, Schott glass) at 4000 rpm for 30 s, prebaked at 125 °C for 60 s. The coated glass was then exposed through a photomask, developed in AZ 300 MIF (Clariant, U.S.A.) for 30 s, and then rinsed in deionized water and dried with nitrogen gas. After lithographic patterning, 100 nm thick Ti and 300 nm thick Pt layers were deposited by advanced magnetron sputtering techniques, and the undeveloped photoresist with unwanted metal coating was then removed by “lift-off” with acetone. To insulate the conducting lines and expose the circular microelectrodes area, a thin layer of SU-8 2002 (Microchem, Newton, MA, U.S.A.) negative photoresist was spin-coated on the Pt MEA, prebaked at 65 °C for 1 min and 95 °C for 2 min. The coated glass was exposed through the designed insulating mask using a mask aligner. Following the exposure, the glass substrate was postbaked at 65 °C for 1 min and 95 °C for 2 min, developed in SU-8 developer, and was then rinsed with isopropyl alcohol and dried with nitrogen gas. The Pt/Ti MEAs chip were electrochemically characterized by using K3[Fe(CN)6] and dopamine. A cleaning protocol by acetone, absolute ethyl alcohol, ultrapure water (Millipore, 18.2 MΩ 3 cm), and 2 mol/L nitric acid solution successively was applied to deal with the MEA before and after detection. The amperometric responses to DA by Pt/Ti microelectrodes on the MEA chip were calibrated by DA standard solutions. Electrochemical experiments were performed by using a CHI 660a electrochemical workstation (CH instruments Inc.) in a threeelectrode arrangement including Pt/Ti working electrode, Pt counter electrode, and Ag/AgCl reference electrode. All the electrochemical experiments were performed in the Faraday cage at room temperature (25 °C). PDMS Microfluidic Chip for Continuous Perfusion Culture of Cells. The upper double layer of the PDMS chip was fabricated using soft-lithography technology37,38 with the bottom one (Figure 1d) for medium perfusion and cell culture and upper one (Figure 1e) for cells and perfusion medium inlet and waste outlet. The two masters were fabricated by standard photolithography techniques. The master for bottom layer was obtained by patterning two layers of photoresists (SU-8 2002 negative photoresist for making the 2 μm height perfusion channel and pillars and AZ 50XT positive photoresist for the 50 μm height circular “moat” and cell culture chamber, Figure 1f) on a silicon wafer. The master for the upper layer was obtained by patterning AZ 50XT photoresist on another silicon wafer. After the silicon masters were exposed to trimethylchlorosilane for 1 min, the mixed PDMS (RTV615, GE Toshiba Silicones Co. Ltd.) prepolymer (polymer/curing agent mass ratio of 10:1) was cast onto 9525

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Figure 1. Layered structure of the multilayer microdevice: (a) glass slide with Ti/Pt MEA, (b) 2 μm thick SU-8 2002 photoresist insulator, (c) 20 μm thick PDMS layer, (d) PDMS layer with cell culture chamber and medium perfusion channel, (e) PDMS layer with cell loading channel and medium/ waste reservoir, (f) high aspect ratio circular “moat” and PDMS micropillars structure around the cell culture chamber, (g) schematic picture of the integrated device, and (h) photograph of the real integrated microdevice.

the top layer, degassed, and cured at 75 °C for 1 h, then peeled off the master mold, followed by punching 3.5 mm in diameter cell loading and waste solution out holes. The thin bottom layer (100 μm height) was achieved by spin-coating PDMS prepolymer at 700 rpm for 30 s and followed by curing at 75 °C for 1 h. The chamber for cell culture was punched by a 0.9 mm diameter syringe needle; after that the cured PDMS peeled off from the master mold. The two PDMS layers were then irreversibly bonded under a upright microscope. Finally, the assembly was irreversibly bonded to a plane or MEA coverslip (Figure 1, parts g and h) for cell culture or amperometric detection experiments. To promote the bonding between MEA and the microfluidic device, a 20 μm thickness PDMS layer was spin-coated onto the insulated MEA, and a 0.9 mm diameter hole was then drilled before being aligned and bonded to PDMS cell culture chip. Continuous Perfusion Culture of Cells. The medium mass transfer (fluid flow) from inlet to cell culture chamber through the circular “moat” and micropillars was visualized using FITC under a confocal microscopy (Andor Revolution, U.K.), and the mass transfer simulation was done using the COMSOL software package (COMSOL Multiphysics v3.5a, Burlington, MA). Briefly, time-dependent mass transfer simulation was achieved by the transient analysis of incompressible Navier
Stockes with species transport of the microfluidics module. The entire domain was meshed using tetrahedron finite elements (totally 397 134 elements). The perfusion effect is characterized by FITC florescence solution at the same inlet velocity as cell culture medium perfusion. As the boundary condition, a laminar inflow was set at the inlet, zero pressure in the two outlets, and no slip condition on the walls. The velocity of the culture medium of different parts within the microfluidic cell culture device was characterized by

the velocity field simulation at the inlet, ring shape channel, and cell culture chamber area. The microfluidic chip model used in the simulation has the same structure and size with the real ones used in experiment. ECV304 cells were maintained in medium RPMI 1640 supplemented with 12% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, and 2% glutamine and were propagated in a humidified incubator at 37 °C with 5% CO2. The pheochromocytoma tumor cell line (PC12) was cultured at 37 °C with 5% CO2 in RPMI 1640 containing 10% fetal bovine serum, 5% horse serum (Invitrogen), 2 mM L-glutamine, and 10 mM NaHCO3. Cells used in the experiments were cultured for 3 days and then suspended in fresh medium at the density of 1.2  106 cells/mL. Microfluidics were sterilized with 75% (v/v) aqueous ethanol for 30 min and then with a 45 min of exposure to UV light in a clean hood in sequence. The elastic tubing and metal pipe were also sterilized under UV light before use, which interfaced the microfluidics with the syringe pump for fluid transport. After sterilization, the culture chamber was coated with 0.01% PLL solution for 1 h and then washed with fresh RPMI 1640 medium. Fresh RPMI 1640 medium was then loaded. After steady fluidic velocity was achieved, cell suspension was added into the sampling chamber, and cells were then moved gently to the culture chamber along the channel. Excessive cells in the sampling chamber were removed, and after 2 h for cell adherence, RPMI 1640 medium was added into the perfusion inlet for continuous perfusion culture at a moderate flow rate in a CO2 incubator (Heracell 150i, Thermo Scientific, U.S.A.). In the PC12 differentiation experiment, a sterilized chip was coated by 10 μg/mL laminin for 3 h to be beneficial to PC12 cells adherence and axon 9526

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was cleaned with ultrapure water and dried by N2 for the next use.

’ RESULTS AND DISCUSSION

Figure 2. Bright-field microscope photograph (a) and electrochemical characterization of the Pt/Ti microelectrode on the MEA chip (b
d): (b) cyclic voltammetric curve obtained from a Pt/Ti microelectrode (25 μm in diameter) in 0.1 mM dopamine (DA); (c) calibration curve for DA over the range of interest; (d) cyclic voltammetric curves before (1) and after cell experiments (2) and after refresh (3) in 5 mM K3[Fe(CN)6]. In panel a the scale bar represents 100 μm.

growth, and DMEM/F12 medium containing 100 ng/mL NGF was added into the perfusion inlet to promote the neuronal differentiation. During cell culture, the cell proliferation and differentiation were observed in real time by an automated live cell imaging system (AxioObserver Z1 fluorescent microscope with camera and incubation system, Zeiss, Germany). Real-Time Amperometric Detection of Dopamine Release via Exocytosis from Cultured PC12 Cells. After culturing for a certain period (1
4 days), the cell culture medium was replaced by phosphate-buffered saline (PBS) (pH = 7.4), keeping the cell culture system in a humidified atmosphere at 37 °C with 5% CO2 for 30 min to let the cell recover. Electrochemical monitoring of PC12 cells was performed by using patch clamp amplifier (HEKA, EPC 10) at room temperature on the stage of an inverted microscope (Axiovert 200M, Zeiss, G€ottingen, Germany) placed in a Faraday cage. Stimulant high K+ solution (400 mM) was injected into the cell culture chamber containing adherent PC12 cells via the medium perfusion channel, which provoked a fast modification of the cell membrane and induced its depolarization and to activate release of intracellular dopamine. Release events were monitored in real time by amperometry at a constant potential of 650 mV versus a Ag/AgCl reference electrode fixed into the chamber simultaneously by independently addressable Pt/Ti microelectrodes. Signals were sampled at 20 kHz, besselfiltered at 2.9 Hz, and digitally filtered at 1 kHz. Raw amperometric data were collected using “Patch Master” and then analyzed by Igor Pro (Igor Pro, Wave Metrics) and minianalysis 6.0 software; only events larger than 5-fold the rms (root-meansquare) noise were used for analysis. After detection, PC12 cells were broken and washed away using ultrapure water for 30 min. The IAMEA was ultrasonically washed successively by acetone, absolute ethyl alcohol, and ultrapure water followed by activation in 2 mol/L HNO3 aqueous solution for 30 min. Then, the integrated microdevice

Electrochemical Characterizations of the MEA. Electrochemical behaviors of the MEA were characterized, and both the cyclic voltammetric and amperometric detection of dopamine (DA) showed that the sputtered Pt/Ti microelectrodes had a favorable and fast electrochemical response (Figure 2b). The limiting current is 0.40 nA, which is in agreement with the theoretical value (0.48 nA) calculated by the equation ilim = 4nFDCr, where n is the number of electrons (2 in this case), F is Faraday’s constant, D is the diffusion coefficient (∼5.0  10
6 cm2 s
1 for DA), C is the analyte concentration (0.1 mM), and r is the radius of the electrode (∼12.5 μm). Concerning the stability of MEA for detection of cultured cells, the electrochemical response quality was checked after a thin layer of PLL coating was deposited on the electrodes, and the averaged electrochemical response was observed to decrease only by 20% compared with uncoated ones (n = 10). The PLL-coated MEA was amperometrically calibrated by DA standard solutions before cell experiments, and an excellent linear amperometric response was obtained over the DA concentration range from 1 to 100 μM (Figure 2c). Furthermore, a cleansing protocol has been developed using HNO3 solution to get rid of the proteins adsorbed on the surface of electrodes after cell culture and monitoring experiments. The electrochemical signal could thus be restored to the original value after cleaning, which facilitated the repeat use of this device (Figure 2d). Mass Transfer in the Integrated Multilayer Microdevice. The ability to control the microenvironment is essential for cell culture in vitro and promoting cell biology and biomedical research. Before coupling to MEA, the double-layer PDMS microfluidic cell culture system was bonded to a glass slide, and its capabilities in automated long-term cell perfusion culture including PLL/laminin coating, cell loading/patterning, and medium perfusion operations were investigated. To offer a stable and uniform microenvironment and fresh medium for cell culture, a combination of a high aspect ratio design and “bottom-up” perfusion model was developed (Figure 1f). After the cells were introduced into the culture chamber from the upper cell loading inlet, the cell culture medium was then perfused at a moderate flow rate. The fresh medium was first filled up to the outer circular “moat” structure (50 μm in height) around the cell culture chamber, and then flowed through the PDMS micropillars (2 μm in height) before reaching the cell culture chamber placed at a 150 μm height (Supporting Information movie S1). The simulation of fluid flow from the perfusion channel to cell culture chamber is presented in Figure 3a
e, and the results were further verified by flow of fluorescent dye at the corresponding time points (Figure 3f
i). Note that the medium perfusion inlet channel and perfusion outlet channel were imprinted at the bottom and top layer of the double-layer microfluidic device, respectively, which leads to a “bottom-up” perfusion device. The novel “circular-center” and “bottom-up” medium perfusion (Figure 3k) using this high aspect ratio integrated microfluidic device not only provides a very stable and uniform microenvironment for cells and also efficiently diminished the negative effects from the shear force created by running liquid on the cells. At a medium flow rate of 0.2 μL/h, cultured cells were continuously perfused by fresh medium and the medium in the 9527

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Figure 3. Simulation of fluid flow from the perfusion channel (2 μm height) to cell culture chamber (150 μm height) through the high aspect ratio circular “moat” (50 μm height) and micropillars (2 μm height) at different time points at a flow rate of 0.2 μL/h (a
e), which was further verified by flow of fluorescent dye FITC (f
j); simulation of velocities of cell culture medium inside the perfusion channel (l), circular “moat” (m), and cell culture chamber (n). The scale bar represents 100 μm.

Figure 4. ECV304 cells loaded into the cell culture chamber after being cultured and propagated for 1, 2, 3, 4, and 5 days (a
e), and the viability was tested by labeling the cultured cells with calcein-AM and PI (f); the scale bar represents 200 μm.

culture chamber (0.9 mm in diameter and 150 μm in height) was replaced about 1.5 times per hour, which facilitated the removal of waste from cell metabolism. The simulation results on velocities of medium inside the perfusion channel, circular “moat”, and cell culture chamber are displayed in Figure 3, parts l and m. These results showed that the velocity of culture medium inside the cell culture chamber was under 0.2 μm/s, which resulted in a shear force less than 0.1 mdyn/cm2 (5.76  10
5 dyn/cm2). Long-Term Automated Perfusion Culture of Cells. High aspect ratio using the ring-shaped micropillar array surrounding the cell culture chamber in combination with the automated “circular-center” and “bottom-up” perfusion model successfully provided continuous fresh medium in a moderated speed and a stable and uniform environment for cells. Furthermore, during the culturing process, all the manual manipulations which are necessary for traditional cell culture were totally avoided. Cells were patterned on the substrate 2 h after being loaded in the chamber and began to propagate as expected. No cell detachment or floating cell debris was observed during the culture process when the medium perfusion rate was fixed below 5 μL/h. By using this microfluidic device, cells could be cultured for a relatively longer time than traditional methods, and the ECV304

cells have been cultivated and propagated for 6 days (Figure 4 and Supporting Information movie S2). In our experiments, 14 days of culture has been realized using a 2 mm diameter chamber when the density of the initially patterned cell was well-controlled. The viability of the cultured cells was checked by injection of calcein-AM and PI from the perfusion inlet to label cells, and a viability of over 98% was obtained. PC12 cells were also successfully differentiated to neurons after being cultured for about 10 days (Supporting Information Figure S2). Amperometric Monitoring of the Exocytosis from Individual Cultured Cells. Amperometric monitoring of the exocytosis from individual cultured cells in real time was then performed by using the system that integrated the microfluidic perfusion cell culture device and MEAs. The PC12 cells were introduced into the integrated device and cultured according to the above protocol. During electrochemical monitoring, the cell culture medium was replaced by PBS solution and the stimulant was injected to induce the DA release via exocytosis. Figure 5a presents the typical results depicting the amperometric monitoring of DA release from PC12 cells cultured inside this device for 2 days simultaneously detected at three individual patterned Pt microelectrodes. Quantal release events were recorded as clearly resolvable spikes, which is similar to that previously reported with carbon fiber electrode (CFE) amperometry.39,40 Detection of brief spikes with short half-width (2.01 ( 0.06 ms) indicated the excellent electrochemical sensitivity of the patterned electrodes since the released dopamine was rapidly oxidized. Moreover, when MEA was coupled to the microfluidic cell perfusion culture chip, the exocytosis events from a higher percentage of cells could be detected, showing that the microfluidic chip provided a comfortable environment for cells and a better viability. Well-defined amperometric signals could be obtained during the 4 day cell culturing period, while culture time longer than 4 days resulted in a decline of the amplitude and frequency of amperometric spikes, possibly due to the adsorption of secreted proteins from cells which decreased the electrodes’ sensitivity. 9528

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Figure 5. Amperometric recordings from three electrodes recorded from three single PC12 cells culture in the microdevice for 2 days stimulated by high K+ solution; the asterisk-labeled spike is amplified in the inset (a). Histograms of width at half-height of events (b), peak currents (c), and released molecules (d) obtained from amperometric recordings.

Table 1. Mean Values ((SE) of Quantitative and Kinetic Parameters of Amperometric Spikes of the Exocytotic Events Detected (n = 596) Imax [pA]

tspikes [ms]

Q [fC]

N [ 106 molecules]

183.69 ( 8.76

2.01 ( 0.06

600.99 ( 27.23

1.88 ( 0.85

The statistics on the kinetic parameters from the amperometric spikes are presented in Table 1 and Figure 5b
d. The half-width of the spikes displayed a narrow (concentrated) distribution resulted from tight adherence of the base of the cells (basal) to patterned electrode surface (Figure 5b). In this electrode
cell arrangement, the time course accounting for the diffusion of dopamine from release sites to the electrode could be ignored, and the exocytotic kinetics at the basal pole could be herewith precisely investigated. However, the mean values of Imax (183.69 ( 8.76 pA) and Q (600.99 ( 27.23 fC) are higher than what are usually reported using CFE, and has a similar trend with several observations recently reported.11,29,41,42 Though the matter cannot be solved unambiguously at this stage, we think several hypotheses may help to rationalize our results based on the literatures. First, the cells were cultured and tightly adhered on the microelectrode with diameter of 25 μm, and a larger fraction of released molecules could be measured compared to using CFME with the diameter of 5
7 μm on the top of the cells. Second, the polarized nature of the cell and the intrinsic differences exist between the basal pole and apical pole owing to the local membrane dynamics difference.41 Lastly, several groups reported that homotypic fusion by the mean of prefusion of vesicles at the base of the cell before exocytosis occurred, which usually led to massive events;42
44 therefore, another possible explanation for the huge amperometric spikes is that a higher percentage homotypic fusion of vesicles occurred at the basal pole of the PC12 cells during the culture process.

’ CONCLUSIONS MEA provides a high-throughput, labor-free, and automated or semiautomated technique for electrochemical detection on living cells. Microfluidic perfusion culture allows one to deliver a background of soluble factors as well as constantly changes the nutrients, etc., so as to mimic more accurately in vivo situations. In this paper, a multifunctional microfluidic platform which integrated a high aspect ratio PDMS microfluidic cell perfusion culture device and an IAMEA for electrochemical monitoring of cells was developed, and the method proposed here successfully permitted monitoring of exocytosis during cell growth and differentiation for the first time. The integration of the microfluidic system with MEA provides the capability for real-time monitoring of cells during their physiological process. The cell behaviors at different cell growth and development phases can therefore be investigated in situ and in real time. Furthermore, with the use of microfluidics, each cell as well as extracellular medium and other affecting factors can be controlled in a uniform or distinct way. Therefore, the present strategy provided a valuable entry for the accurate monitoring of single cells or cellular communication networks under precisely controlled cellular microenvironments. Future improvements involve the microfabrication of a novel contamination-free or self-cleaning electrode for longtime detection. These two aspects are currently in progress in our laboratory. ’ ASSOCIATED CONTENT

bS

Supporting Information. Fabrication process for MEA (Figure S1), differentiation of PC12 cells (Figure S2), fluid flow of FITC into the cell culture chamber (movie S1), culture of ECV304 cells during two days (movie S2). This material is available free of charge via the Internet at http:// pubs.acs.org.

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’ AUTHOR INFORMATION Corresponding Author

*Phone: (86)2768752149. Fax: (86)2768754067. E-mail: [email protected].

’ ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (Nos. 20975077, 31070995), the Science Fund for Creative Research Groups (No. 20921062), the National Basic Research Program of China (973 Program, No. 2007CB714507), the Program for New Century Excellent Talents in University (NCET-10-0611), and the Program for Changjiang Scholars and Innovative Research Team in University (IRT1030). We thank Professor Christian Amatore for helpful suggestions and discussions. The first two authors contributed equally to this work. ’ REFERENCES (1) Wightman, R. M.; Jankowski, J. A.; Kennedy, R. T.; Kawagoe, K. T.; Schroeder, T. J.; Leszczyszyn, D. J.; Near, J. A.; Diliberto, E. J.; Viveros, O. H. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 10754–10758. (2) Mosharov, E. V.; Sulzer, D. Nat. Methods 2005, 2, 651–658. (3) Wightman, R. M. Science 2006, 311, 1570–1574. (4) Schulte, A.; Schuhmann, W. Angew. Chem., Int. Ed. 2007, 46, 8760–8777. (5) Amatore, C.; Arbault, S.; Guille, M.; Lema^itre, F. Chem. Rev. 2008, 108, 2585–2621. (6) Wu, W. Z.; Huang, W. H.; Wang, W.; Wang, Z. L.; Cheng, J. K.; Xu, T.; Zhang, R. Y.; Chen, Y.; Liu, J. J. Am. Chem. Soc. 2005, 127, 8914–8915. (7) Lin, Y.; Trouillon, R.; Safina, G.; Ewing, A. G. Anal. Chem. 2011, 83, 4369–4392. (8) Huang, Y.; Cai, D.; Chen, P. Anal. Chem. 2011, 83, 4393–4406. (9) Wang, W.; Zhang, S. H.; Li, L. M.; Wang, Z. L.; Cheng, J. K.; Huang, W. H. Anal. Bioanal. Chem. 2009, 394, 17–32. (10) Bratten, C. D. T.; Cobbold, P. H.; Cooper, J. M. Anal. Chem. 1998, 70, 1164–1170. (11) Chen, P.; Xu, B.; Tokranova, N.; Feng, X.; Castracane, J.; Gillis, K. D. Anal. Chem. 2003, 75, 518–524. (12) Cui, H. F.; Ye, J. S.; Chen, Y.; Chong, S. C.; Sheu, F. S. Anal. Chem. 2006, 78, 6347–6355. (13) Sp
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