Electrophoretic Cell Manipulation and Electrochemical Gene-Function

Mar 26, 2008 - To whom correspondence should be addressed. ... chambers was developed in order to perform single-cell-based gene-function analysis...
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Anal. Chem. 2008, 80, 3722–3727

Electrophoretic Cell Manipulation and Electrochemical Gene-Function Analysis Based on a Yeast Two-Hybrid System in a Microfluidic Device Tomoyuki Yasukawa,*,† Kuniaki Nagamine,† Yoshiko Horiguchi,† Hitoshi Shiku,† Masahiro Koide,‡ Tomoaki Itayama,‡ Fujio Shiraishi,§ and Tomokazu Matsue*,† Graduate School of Environmental Studies, Tohoku University; 6-6-11-604, Aramaki-Aoba, Aoba, Sendai 980-8579 Japan, and Environmental Chemistry Division and Research Center for Environmental Risk, National Institute for Environmental Studies, 16-2, Onogawa, Tsukuba 305-8506, Japan A novel microfluidic device with an array of analytical chambers was developed in order to perform single-cellbased gene-function analysis. A series of analytical processes was carried out using the device, including electrophoretic manipulation of single cells and electrochemical measurement of gene function. A poly(dimethylsiloxane) microstructure with a microfluidic channel (150 µm in width, 10 µm in height) and an analytical chamber (100 × 20 × 10 µm3) were fabricated and aligned on a glass substrate with an array of Au microelectrodes. Two microelectrodes positioned in the analytical chamber were employed as a working electrode for the electrophoretic manipulation of cells and electrochemical measurements. A yeast strain (Saccharomyces cerevisiae Y190) carrying the β-galactosidase reporter gene was used to demonstrate that the device could detect the enzyme. Target cells flowing through the main channel were introduced into the chamber by electrophoresis using the ground electrode laid on the main channel. When the cell was treated with 17β-estradiol, gene expression was triggered to produce β-galactosidase, catalyzing the hydrolysis of p-aminophenyl-β-D-galactopyranoside to form p-aminophenol (PAP). The enzymatically generated PAP was detected by cyclic voltammetry and amperometry at the single-cell level in the chamber of the device. Generatorcollector mode amperometry was also applied to amplify the current response originating from gene expression in the trapped single cells. After electrochemical measurement, the trapped cells were easily released from the chamber using electrophoretic force. In the postgenome era, rapid and high-throughput screening methods have become indispensable for genomewide genefunction analysis. Large-scale recombinant cell-based assays have * To whom correspondence should be addressed. Tomoyuki Yasukawa: (current address) Graduate School of Material Science, University of Hyogo, 3-2-1 Kouto, Kamigori-cho, Ako-gun, Hyogo 678-1297 Japan. E-mail: yasu@ sci.u-hyogo.ac.jp. Phone: +81-791-58-0171. Fax: +81-791-58-0493. Tomokazu Matsue: Graduate School of Environmental Studies, Tohoku University, 6-6-11604, Aramaki-Aoba, Aoba-Ku, Sendai 980-8579, Japan. E-mail: matsue@ bioinfo.che.tohoku.ac.jp. Phone and fax: +81-22-795-7209. † Tohoku University. ‡ Environmental Chemistry Division, National Institute for Environmental Studies. § Research Center for Environmental Risk, National Institute for Environmental Studies.

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been performed to investigate gene functions in living cells.1,2 Cellular responses originating from gene expression have been characterized using several approaches, such as monitoring transcriptional and translational regulation with reporter genes and monitoring the respiratory activity of cells,3–5 observing subcellular distribution of proteins with a reporter protein tagged to the target protein,6,7 and detecting protein–protein interactions based on a two-hybrid system.8,9 The genome DNA expression library of recombinant living cells is conventionally screened using an isolated colony array format on an agar plate or a microtiter plate in which each colony expresses a defined clone. Conventional protocol requires repeated isolation procedures to obtain single colonies or clones due to slow growth in a selection medium including antibiotics or limited nutrients. Therefore, because of the complicated procedures and human observation of colony formation, human error is a major source of precision and efficiency loss. To progress in genomewide gene-function analysis, a rapid and precise method for isolating single cells and a highly sensitive system to detect single cellular responses are required. An integrated microfluidic device creates the opportunity to analyze single cells in bulk. A series of single-cell-based assays has been performed using a single microdevice,10 for example, cell culture and stimulation by controlling the microculture environment through the microchannel, biological analysis of cellular responses reflecting gene function, selective isolation of a single cell from a heterogeneous cell mixture depending on cellular responses, and molecular biological analysis of cell lysate (1) Ziauddin, J.; Sabatini, D. M. Nature 2001, 411, 107–110. (2) Bailey, S. N.; Ali, S. M.; Carpenter, A. E.; Higgins, C. O.; Sabatini, D. M. Nature Methods 2006, 3, 117–122. (3) Van Dyk, T. K.; DeRose, E. J.; Gonye, G. E. J. Bacteriol. 2001, 183, 5496– 5505. (4) Kuang, Y.; Biran, I.; Walt, D. R. Anal. Chem. 2004, 76, 2902–2909. (5) Nagamine, K.; Matsui, N.; Kaya, T.; Yasukawa, T.; Shiku, H.; Nakayama, T.; Nishino, T.; Matsue, T. Biosens. Bioelectron. 2005, 21, 145–151. (6) Kitagawa, M.; Ara, T.; Arifuzzanman, M.; Ioka, T.; Inamoto, E.; Toyonaga, H.; Mori, H. DNA Res. 2005, 12, 291–299. (7) Phillips, D. R. FEMS Microbiol. Lett. 2001, 204, 9–18. (8) Uetz, P.; Glot, L.; Cagney, G.; Mansfield, T. A.; Judson, R. S.; Knight, J. R.; Lockshon, D.; Narayan, V.; Srinivasan, M.; Pochart, P.; Qureshi-Emili, A.; Li, Y.; Godwin, B.; Conover, D.; Kalbfleish, T.; Vijayadamodar, G.; Yang, M.; Johnston, M.; Fields, S.; Rothberg, J. M. Nature 2000, 403, 623–627. (9) Biran, I.; Walt, D. R. Anal. Chem. 2002, 74, 3046–3054. (10) El-Ali, J.; Sorger, P. K.; Jensen, K. F. Nature 2006, 442, 403–410. 10.1021/ac800143t CCC: $40.75  2008 American Chemical Society Published on Web 03/26/2008

by extracting DNA, mRNA, protein, or a small molecule from the cell. Furthermore, rapid and quantitative analysis of the cell can be performed with an integrated microfluidic device, which eliminates the need for intensive labor and decreases the cost of biological experiments. We have developed a microfluidic device for single recombinant cell-based gene-function analysis. A series of analytical processes were carried out, including electrophoretic isolation of single cells, electrochemical measurement of gene function, and electrophoretic collection of the desired single cell from the microfluidic device. We have performed an electrochemical evaluation of protein expression resulting from a protein–protein interaction in single yeast cells using this device. The strain of yeast we used (Y190) has been used for the estrogenicity assay of chemical compounds based on the two-hybrid system,11 wherein the estrogenic ligand-dependent interaction between a hormone receptor and a coactivator is detected by the expression of β-galactosidase using a reporter protein. Huge numbers of chemicals have been tested for estrogenic activity to screen suspected endocrine disruptors in this way.11,12 In this study, we detected β-galactosidase expression induced by a steroidal hormone, 17β-estradiol, to demonstrate the performance of the electrochemical microfluidic device for monitoring protein expression in a single cell. MATERIALS AND METHODS Reagents. Primer (hexamethyl disilazane) and positive photoresist (S1805, S1818) were purchased from Shipley Far East Ltd., Japan. SU-8 2010 was purchased from Microchem Co. Poly(dimethylsiloxane) (PDMS; Sylgard 184) was purchased from Dow Corning, Co. 17β-Estradiol was purchased from Sigma. p-Aminophenyl-β-D-galactopyranoside (PAPG) was purchased from Tokyo Chemical Industry Co., Ltd., Japan. Triton X-100 was purchased from Polysciences, Inc. Dimethyl sulfoxide (DMSO), Na2HPO4 · 12H2O, NaH2PO4 · 2H2O, KCl, and p-aminophenol were purchased from Wako Pure Chemicals, Japan. MgSO4 · 7H2O was purchased from Kanto Chemical Co., Inc., Japan. 2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) was purchased from DOJINDO, Japan. All solutions were prepared using distilled and deionized water from Direct-Q (Millipore). Fabrication of the Microfluidic Device for Cell Manipulation and Electrochemical Measurement. Au or Pt microelectrode arrays were fabricated on a quartz glass substrate using conventional photolithography techniques. The quartz substrate was cleaned with ultrasonication in acetone and 2-propanol and then dried with nitrogen gas. A primer and positive photoresist (S1805) was spin-coated at 3000 rpm for 20 s on the quartz substrate, and the substrate was baked at 95 °C for 10 min. The substrate was irradiated using a UV light for 8 s through a chromium mask with microelectrode patterns. After developing with developer, Ti and Au were deposited on the substrate by sputtering to create a Ti/Au multilayer (100 nm thick). The electrode pattern was revealed using a lift-off technique by immersing the electrode substrate in an acetone bath. (11) Nishihara, T.; Nishikawa, J.; Kanayama, T.; Dakeyama, F.; Saito, K.; Imagawa, M.; Takatori, S.; Kitagawa, Y.; Hori, S.; Utsumi, H. J. Health Sci. 2000, 46, 282–298. (12) Shiraishi, F.;Shiraishi, H.;Nishikawa, J.;Nishihara, T.;Morita, M. J. Environ. Chem. 2000. 10, 57–64 (in Japanese).

Figure 1. (A) Schematic cross-sectional view of the configuration of the analytical chamber included principles behind the enzymatic reaction and the electrochemical detection. (B) Optical image of the whole microfluidic device with an array of four analytical chambers. (C) Principles of (C-1 and C-3) the manipulation of cells and (C-2) the electrochemical measurement of expressed enzyme in the microfluidic device.

The Ti/Au lead patterns were insulated by a SiO2 layer (150 nm thick) by sputter deposition, and an electrochemically active window exposed to the solution was defined by lift-off lithography using a positive photoresist (S1818) (Figure 1A). The resulting substrates were annealed for 1 h at 800 °C to improve the insulation property of the SiO2 layer. The PDMS microfluidic channels and analytical chambers were fabricated by curing the prepolymer on a glass substrate with a master. The master was photolithographically patterned using a negative photoresist (SU-8 2010). A 10:1 mixture of silicon elastomer and the curing agent was poured on the master and left at 100 °C for 1 h to cure the prepolymer. The PDMS replica was then peeled from the glass substrate and cut to an appropriate size. Vertical holes for the reservoir were made at both ends of the microchannel using a needle. Finally, the substrate with microelectrodes and the PDMS microchannel were combined to create a microfluidic device with electrodes. Analytical Chemistry, Vol. 80, No. 10, May 15, 2008

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The whole microfluidic device with an array of four analytical chambers is shown in Figure 1B (see Figure S1 in Supporting Information for the magnified pictures). A band electrode (50 µm in width) is located in the middle of the main channel (length 20 mm, width 150 µm, height 10 µm) and two microelectrodes, E1 and E2, are located in grooved analytical chambers (length 100 µm, width 20 µm, height 10 µm) to the side of the main channel. The band electrode was used as a ground in order to guide the cell into and from the analytical chamber and also acted as a counter electrode for electrochemistry. E1 and E2, positioned in the chamber, were used as working electrodes to electrophoretically manipulate the cell and electrochemically characterize its enzyme activity. The geometric area exposed to the solution was 10 × 10 µm2 for E1 and 10 × 20 µm2 for E2. The gap between E1 and E2 was set at 10 µm. Four analytical chambers were prepared in the channel, 2.0–4.0 mm apart. Yeast Strain and Growth Conditions. The yeast strain Saccharomyces cerevisiae Y190 was obtained from Clontech. The expression plasmids for medaka estrogen receptor R (medERR: Oryzias latipes) fused to the GAL4 DNA binding domain (GAL4 DBD) and coactivator TIF2 fused to the GAL4 activation domain (GAL4 AD) were introduced into yeast cells carrying the β-galactosidase reporter gene.11,12 The cells were preincubated for 24 h at 30 °C with shaking at 100 rpm in modified SD medium (without tryptophan and leucine). The culture (250 µL) was then mixed with medium (250 µL) containing 20 nM 17β-estradiol and 2.0% (v/v) DMSO and incubated for 4 h at 30 °C with shaking at 100 rpm to induce β-galactosidase expression. An amount of 333 µL of Z buffer (60.0 mM Na2HPO4 · 12H2O, 39.7 mM NaH2PO4 · 2H2O, 10.0 mM KCl, 10.0 mM MgSO4 · 7H2O, pH 7.0) including 0.75% (v/v) Triton X-100, a nonionic surfactant, was added to the culture and incubated for 1 h at 30 °C with shaking at 100 rpm in order to improve the permeability of the yeast cell membrane.13 The yeast cells were collected by centrifugation (6000 rpm for 5 min), washed three times with 20 mM HEPES buffer solution (pH 7.0), and then resuspended in 300 µL of 20 mM HEPES buffer containing 0.3% Triton X-100 (final concentration of yeast cells was 1 × 107 cells/mL) for the following procedures. Electrophoretic Manipulation of a Single Yeast Cell in the Microfluidic Channel. The behavior of electrophoretically manipulated yeast cells in the microchannel was observed through a microscope equipped with a digital CCD camera and a video monitor. Figure 1C shows the principle of the manipulation of cells and the electrochemical measurement of expressed enzyme in the microfluidic device. A suspension of yeast cells dispersed in 20 mM HEPES buffer solution containing 0.3% Triton X-100 was injected into the channel, and the flow rate was regulated by a syringe pump that was set at 40 µm/s. When one of the flowing cells approached the chamber opening, +3.0 V dc voltage was applied to E1 against the ground electrode in the main channel using a function generator in order to introduce the cell into the chamber (Figure 1C-1). To avoid long-term application of the electric field to the trapped yeast cells, the dc voltage was immediately turned off after the cells were positioned in the chamber. The trapped cells were released from the chamber by applying -3.0 V dc voltage to E1 or E2 against the ground electrode (Figure 1C-3). (13) Chow, C. K.; Palecek, S. P. Biotechnol. Prog. 2004, 20, 449–456.

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Equipment and Electrochemical Detection of β-Galactosidase Activity in Yeast Cells. Electrochemical signals in the analytical chamber were detected using a potentiostat (HA1010 mM8; Hokuto Denko Corp., Tokyo, Japan) with an Ag/AgClsaturated KCl electrode inserted into the inlet of the main channel as the reference electrode. All measurements were done at room temperature in a Faraday cage. After the yeast cells were trapped in the analytical chamber, the solution in the channel was completely exchanged with Z buffer containing 7.4 mM PAPG and 0.3% Triton X-100 (Figure 1C-2). β-Galactosidase activity expressed in yeast cells was monitored by detecting the oxidation current of p-aminophenol (PAP), a product of the enzyme-catalyzed hydrolysis of PAPG inside the trapped cells (Figure 1A).14–16 Cyclic voltammetry (CV), differential pulse voltammetry (DPV), and potential step amperometry were employed to detect the oxidation current of PAP. The scan rate for CV was set at 50 mV/ s. Potential step amperometry was carried out in both single mode and generator-collector mode, where the potential of the microelectrode E1 in the chamber was stepped to +0.3 V from 0.0 V to detect the oxidation current of PAP, while the potential of the other microelectrode (E2) was held at 0.0 V to reduce quinone imine (QI) generated at E1 (Figure 1A). RESULTS AND DISCUSSION Electrophoretic Manipulation of a Single Yeast Cell in the Microchannel. The microfluidic device was employed in order to manipulate and guide flowing cells into the analytical chambers fabricated aside the main channel. Figure 2 shows sequential optical microscopic images during electrophoretic manipulation of a single yeast cell flowing at 40 µm/s in the main channel (Figure 2A,B). When a dc voltage (+3.0 V) was applied to the working electrode E1 against the ground electrode in the main channel, a single yeast cell near the entrance of the analytical chamber was quickly introduced and trapped deep in the chamber (Figure 2B-D and movie, Supporting Information). The maximum velocity of yeast cells in the analytical chamber was found to be as high as 600 µm/s under these conditions. This fact indicates that the membrane of yeast cells treated with Triton X-100 was negatively charged at physiological pH. The cells remained in the chamber even after switching off the dc voltage, although the solution continued to flow in the main channel. As described below, the activity of the trapped single cells was estimated by generator-collector mode amperometry. Under the experimental conditions, a voltage of 3.0 V was sufficient for the electrophoretic manipulation of yeast cells. In addition, the cells flowing in the main channel did not enter the analytical chamber without the electrophoretic force because there was no fluidic flow in the analytical chamber. The trapped cells were released from the analytical chamber to the main channel by using the electrophoretic repulsive force. When a voltage of –3.0 V was applied to E1, the trapped single cells were directed to the main channel and flowed out downstream (Figure 2E-H and movie, Supporting Information). (14) Scott, D. L.; Ramanathan, S.; Shi, W. P.; Rosen, B. P.; Daunert, S. Anal. Chem. 1997, 69, 16–20. (15) Brian, I.; Klimentiy, L.; Hengge-Aronis, R.; Ron, E. Z.; Rishpon, J. Microbiology 1999, 145, 2129–2133. (16) Masson, M.; Liu, Z.; Haruyama, T.; Kobatake, E.; Ikariyama, Y.; Aizawa, M. Anal. Chim. Acta 1995, 304, 353–359.

Figure 3. (a) Cyclic voltammogram of PAPG and PAP generated from a trapped single cell precultured with 20 nM 17β-estradiol, obtained 30 min after the injection of PAPG. (b) Cyclic voltammogram obtained from 50 cells, which had not been precultivated with 17βestradiol, trapped in the chamber.

Figure 2. (A-D) Sequential optical microscopic images taken during electrophoretic manipulation of a single yeast cell flowing at 40 µm/s in the main channel, from the main channel to the analytical chamber. (E-H) Sequential optical microscopic images for the release of trapped cells from the analytical chamber to the main channel using the electrophoretic repulsive force.

Electrochemical Performance of PAP. In order to detect PAP generated by the enzyme reaction of β-galactosidase expressed in yeast cells, the electrochemical reaction of PAP and PAPG, the β-galactosidase substrate, was investigated by DPV (see Figure S2, Supporting Information). The oxidation peaks for PAP and PAPG were observed separately. Therefore, PAP can be identified by its peak potential in a solution containing PAPG. CV was also performed, to characterize the electrochemical behavior of PAP and PAPG, using E1. The oxidation current for PAP increased at 0.1 V and reached a steady-state at 0.4 V. PAPG was not oxidized in the potential range 0.1–0.4 V and oxidized at potentials more positive than 0.5 V. On the basis of these results, we used amperometry at 0.30 V, as well as DPV, to selectively detect PAP released from yeast cells trapped in the analytical chamber.17–21 Replacement of the Solution in the Analytical Chamber. The activity of β-galactosidase expressed in the trapped yeast cells was estimated in a solution containing 7.4 mM PAPG and 0.3%

Triton X-100. Diffusion of PAPG into the deep analytical chamber affects the electrochemical measurements, so the replacement of the solution in the analytical chamber was investigated by CV (Figure S3, Supporting Information) and amperometry (Figure S4, Supporting Information). When the chamber was filled with the 7.4 mM PAPG solution, a clear oxidation current of PAPG was observed in CV (Figure S3a, Supporting Information). Then, the Z buffer solution with no PAPG was injected. After 3 min of injection, the oxidation current disappeared from the CV (Figure S3b, Supporting Information). The oxidation current for PAPG recovered in the CV 3 min after the reinjection of 7.4 mM PAPG solution (Figure S3c, Supporting Information). The amperometric measurements also suggested that the solution inside the analytical chamber was replaced with the outside solution in the main channel (Figure S4, Supporting Information). A 7.4 mM PAPG solution was injected into the device filled with Z buffer at the point indicated by an arrow. The oxidation of PAPG in the chamber was continuously monitored by amperometry at 0.7 V. The oxidation current of PAPG was observed to increase immediately after the injection of PAPG and reached a steady state within 40 s. These measurements suggested that the solution inside the analytical chamber was replaced within 40 s. Single-Cell-Based Gene-Function Analysis with the Fluidic Device. Hydrolysis of PAPG, catalyzed by β-galactosidase expressed in a single yeast cell, generates PAP which can be detected by DVP in the analytical chamber. Figure 3 shows the cyclic voltammograms observed for a single cell trapped inside the analytical chamber. A single cell precultured with 17β-estradiol was trapped inside the chamber by electrophoretic manipulation, as described earlier, and then a solution containing 7.4 mM PAPG (17) Nagamine, K.; Onodera, S.; Torisawa, Y. S.; Yasukawa, T.; Shiku, H.; Matsue, T. Anal. Chem. 2005, 77, 4278–4281. (18) Nagamine, K.; Onodera, S.; Kurihara, A.; Yasukawa, T.; Shiku, H.; Asano, R.; Kumagai, I.; Matsue, T. Biotechnol. Bioeng. 2007, 96, 1008–1013. (19) Kaya, T.; Nagamine, K.; Matsui, N.; Yasukawa, T.; Shiku, H.; Matsue, T. Chem. Commun. 2004, 2, 248–249. (20) Matsui, N.; Kaya, T.; Nagamine, K.; Yasukawa, T.; Shiku, H.; Matsue, T. Biosens. Bioelectron. 2006, 21, 1202–1209. (21) Cai, L.; Friedman, N.; Xie, X. S. Nature 2006, 440, 358–362.

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Figure 4. Amperograms for PAP oxidation obtained in the analytical chamber with a single cell (a) with and (b) without preculture in 20 nM 17β-estradiol solution. (c) Amperogram obtained in the chamber without cells.

was injected into the channel. Two oxidation current peaks for PAP at 0.22 V and PAPG at 0.67 V were observed 30 min after the injection (Figure 3a), while no peak for PAP was observed from cells that had not been precultured, even when the chamber accommodated as many as 50 cells (Figure 3b). These results clearly indicate that the expression of β-galactosidase from a single yeast cell can be detected using the microfluidic device. The low oxidation peak for Figure 3b was due to the prevention of the diffusion of PAPG from the channel to E1 by cells accommodated in the analytical chamber. Amperometric measurements were also used for single-cellbased gene-function analysis. Figure 4 shows the oxidation currents observed from potential step amperometry (0.0-0.30 V) in the analytical chamber accommodating a single cell (a) with and (b) without preculture in a solution of 20 nM 17β-estradiol. These responses were recorded 30 min after the injection of PAPG to ensure the enzyme reaction had occurred in the single cell. After the spikelike responses due to capacitive effect, the currents decreased and reached almost a steady state. The current response obtained from precultured cells was markedly larger than those without preculture. In addition, the amperometric response obtained from a cell that had not been precultured was at the same level as the background response without cells (Figure 4c). These results suggest that PAP is generated by the enzyme reaction inside the single cell. The generation rate of PAP from the single cell was calculated from the difference of steady-state response between cells with and without preculture and found to be around 1.0 × 10-15 mol/s. Thus, the electrochemical microfluidic device enables highly sensitive measurement of β-galactosidase activity in a single yeast cell. We found that each single cell showed a different electrochemical response. Some cells showed a large response, as shown in Figure 4, but no significant increase was sometimes observed even when a few cells were trapped in the analytical chamber. These differences are due to the different expression levels of individual cells. Biran et al. reported that only 33% of the positivecontrol strains of yeast two-hybrid systems express the model proteins.9 Therefore, a highly sensitive measurement method is desired to detect the different enzyme activities inside the cells. 3726

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Figure 5. Amperograms of 1.0 mM PAP introduced into the chamber in the generator-collector and single modes. (a) Response obtained by the oxidation of PAP at the generator electrode, E1. (b) Response obtained by the reduction of QI at the collector electrode, E2. (c) Response obtained by the oxidation of PAP at E1 without use of E2. E1 was stepped from 0.0 to 0.3 V while E2 was held at 0.0 V.

Figure 6. Amperograms obtained in the analytical chamber with a single cell (A) with and (B) without preculture in 17β-estradiol solution in the generator-collector mode. (a) Response obtained by the oxidation of PAP at the generator electrode, E1. (b) Response obtained by the reduction of QI at the collector electrode, E2.

Generator-collector mode amperometry was applied to detect β-galactosidase activity with high sensitivity. E1 was stepped from 0.0 to 0.3 V, while E2 was held at 0.0 V. Figure 5 shows the amperograms of 1.0 mM PAP introduced into the chamber in the single and generator-collector modes. The amperograms at E1 show spikelike shapes and then reach steady-state currents for oxidation of PAP (Figure 5a). E2 was used to collect and reduce QI generated at E1; the ratio of the reduction current at E2 to the oxidation current at E1 is 79% (Figure 5b). Approximately 20% of QI generated at E1 escaped from the analytical chamber. The oxidation current of PAP in the generator-collector mode is 1.5 times larger than that in the single mode due to the redox cycling between E1 and E2 (Figure 5c). Therefore, in the generatorcollector mode, signal amplification is expected to detect PAP generated from the single cells with high sensitivity. Figure 6 shows the generator-collector mode amperograms in the analytical chamber with a single cell (A) with and (B) without preculture in 17β-estradiol solution. The amperogram shows the distinct oxidation and reduction currents, respectively,

at E1 and E2 when the single cells precultured with 17β-estradiol were trapped in the chamber, indicating redox cycling of PAP/ QI between E1 and E2 (Figure 6A). On the other hand, no change in reduction current response was observed (Figure 6B), although a spikelike capacitive current was observed in the oxidation response at E1. The reduction currents observed at E2 as shown in Figure 6A,b are strong evidence for the generation of PAP by β-galactosidase expressed in the trapped single yeast cells. Thus, our microfluidic device with two adjacent electrodes is a powerful tool for detection of an enzyme expressed at the level of a single cell. CONCLUSIONS The microfluidic device with an array of analytical chambers described here is suitable for the manipulation of single cells and for gene-function analysis at the single-cell level. The cells injected into the main channel were electrophoretically manipulated to be trapped deep in the analytical chamber with a microelectrode (E1) when a dc voltage (+3.0 V) was applied to E1 against the ground electrode in the main channel. The trapped cells remained in the analytical chamber after switching off the voltage but could be released into the main channel by applying voltage with the opposite polarity. Expression of β-galactosidase inside the trapped cell was electrochemically detected at the single-cell level using

this device. Generator-collector mode amperometry was conducted to amplify the electrochemical response based on redox cycling. Further miniaturization and improvement of array systems for analytical chambers will increase the sensitivity of electrochemical detection of PAP and improve the throughput for cell analysis. We believe that an electrochemical microfluidic device of this kind is a powerful tool for detecting cellular responses against various stimuli at the single-cell level. ACKNOWLEDGMENT This work has been financially supported by a Grant-in-Aid for Scientific Research (Grant No. 18101006) from the Ministry of Education, Science and Culture, Japan, and R&D Project for Environmental Nanotechnology from the Ministry of the Environment. K.N. acknowledges support from a research fellowship of the Japan Society for the Promotion of Science. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review January 20, 2008. Accepted February 26, 2008. AC800143T

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