Electrochemical Monitoring of Cellular Signal Transduction with a

Further, we have developed an electrode-integrated cell culture device for parallel evaluation of cellular SEAP expression. The detector electrode was...
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Electrochemical Monitoring of Cellular Signal Transduction with a Secreted Alkaline Phosphatase Reporter System Yu-suke Torisawa, Noriko Ohara, Kuniaki Nagamine, Shigenobu Kasai, Tomoyuki Yasukawa, Hitoshi Shiku,* and Tomokazu Matsue*

Graduate School of Environmental Studies, Tohoku University, Sendai 980-8579, Japan

Electrochemical monitoring of cellular signal transduction under three-dimensional (3-D) cell culture conditions has been demonstrated by combining cell-based microarrays with a secreted alkaline phosphatase (SEAP) reporter system. The cells were genetically engineered to produce SEAP under the control of nuclear factor KB (NFKB) enhancer elements, and they were embedded with a small volume of a collagen gel matrix on a pyramidal-shaped silicon microstructure. Cellular SEAP expression triggered by NFKB activation was assessed by two types of electrochemical systems. First, SEAP expression of a 3-D cell array on a chip was continuously monitored in situ for 2 days by scanning electrochemical microscopy (SECM). Since the SECM-based assay enables the evaluation of cellular respiratory activity, simultaneous measurements of cellular viability and signal transduction were possible. Further, we have developed an electrodeintegrated cell culture device for parallel evaluation of cellular SEAP expression. The detector electrode was integrated around the silicon microhole. Two kinds of cells were immobilized on the array of microholes on the same chip for comparative characterization of their SEAP activity. This electrochemical microdevice can be applied to evaluate the SEAP expression activity in multiple cellular microarrays by a high-throughput method. Cell-based microarrays have recently been developed for rapid and simple evaluation of multiple cellular functions with smallvolume samples.1-5 A lot of research is now focused on the elucidation of gene functions within mammalian cells6 by using genetic screening systems for genomewide analysis in living cells.7-12 Of the various methods available for monitoring gene * Corresponding authors. Fax: +81-22-795-7209. E-mail: shiku@ bioinfo.che.tohoku.ac.jp; [email protected]. (1) Ziauddin, J.; Sabatini, D. M. Nature 2001, 411, 107-110. (2) Mousses, S.; Caplen, N. J.; Cornelison, R.; Weaver, D.; Basik, M.; Hautaniemi, S.; Elkahloun, A. G.; Lotufo, R. A.; Choudary, A.; Dougherty, E. R.; Suh, E.; Kallioniemi, O. Genome Res. 2003, 13, 2341-2347. (3) Revzin, A.; Rajagopalan, P.; Tilles, A. W.; Berthiaume, F.; Yarmush, M. L.; Toner, M. Langmuir 2004, 20, 2999-3005. (4) Anderson, D. G.; Levenberg, S.; Langer, R. Nat. Biotechnol. 2004, 22, 863866. (5) Flaim, C. J.; Chien, S.; Bhatia, S. N. Nat. Methods 2005, 2, 119-125. (6) Wheeler, D. B.; Carpenter, A. E.; Sabatini, D. M. Nat. Genet. 2005, 37, S25-S30. 10.1021/ac060737s CCC: $33.50 Published on Web 10/19/2006

© 2006 American Chemical Society

expression within cells, the reporter gene system is the most commonly used method. To date, several types of proteins have been employed as reporter proteins, e.g., fluorescent protein,13 β-galactosidase,14 and secreted alkaline phosphatase (SEAP).15-19 SEAP is a particularly useful reporter because it possesses a unique feature in that the reporter protein is secreted into the culture medium. The cellular secretion of SEAP is directly proportional to the changes in the intracellular SEAP mRNA.15,16 This characteristic allows the continuous quantification of gene expression. SEAP activity can be sensitively detected by a conventional chemiluminescent or fluorescent assay.17 However, it is difficult to apply it to a high-throughput system using microarrays because such a measurement of SEAP must be performed in the culture medium. We have developed an electrochemical monitoring platform for SEAP reporter assays. Although various cellular microarrays have been proposed, few studies have evaluated cellular functions under three-dimensional (3-D) culture conditions.20 We previously reported a silicon-based biosensor with a 3-D cell culture and applied it to a cellular viability assay.21,22 The 3-D cultured cells were embedded in a small volume (7) Honma, K.; Ochiya, T.; Nagahara, S.; Sano, A.; Yamamoto, H.; Hirai, K.; Aso, Y.; Terada, M. Biochem. Biophys. Res. Commun. 2001, 289, 10751081. (8) Webb, B. L.; Diaz, B.; Martin, G. S.; Lai, F. J. Biomol. Screening 2003, 8, 620-623. (9) Silva, J. M.; Mizuno, H.; Brady, A.; Lucito, R.; Hannon, G. J. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 6548-6552. (10) Yoshikawa, T.; Uchimura, E.; Kishi, M.; Funeriu, D. P.; Miyake, M.; Miyake, J. J. Controlled Release 2004, 96, 227-232. (11) Wheeler, D. B.; Bailey, S. N.; Guertin, D. A.; Carpenter, A. E.; Higgins, C. O.; Sabatini, D. M. Nat. Methods 2004, 1, 127-132. (12) Boutros, M.; Kiger, A. A.; Armknecht, S.; Kerr, K.; Hild, M.; Koch, B.; Haas, S. A.; Consortium, H. F. A.; Paro, R.; Perrimon, N. Science 2004, 303, 832835. (13) Lippincott-Schwartz, J.; Patterson, G. H. Science 2003, 300, 87-91. (14) Rose, M.; Casadaban, M. J.; Botstein, D. Proc. Natl. Acad. Sci. U.S.A. 1981, 78, 2460-2464. (15) Berger, J.; Hauber, J.; Hauber, R.; Geiger, R.; Cullen, B. R. Gene 1988, 66, 1-10. (16) Cullen, B. R., Malim, M. H. Methods Enzymol. 1992, 216, 362-368. (17) Yang, T.-T.; Sinai, P.; Kitts, P. A.; Kain, S. R. BioTechniques 1997, 23, 1110-1114. (18) Kelso, E.; McLean, J.; Cardosi, M. F. Electroanalysis 2000, 12, 490-494. (19) Meng, Y.; Kasai, A.; Hiramatsu, N.; Hayakawa, K.; Takeda, M.; Shimizu, F.; Kawachi, H.; Yao, J.; Kitamura, M. Kidney Int. 2005, 68, 886-893. (20) Sanchez-Bustamante, C. D.; Kelm, J. M.; Mitta, B.; Fussenegger, M. Biotechnol. Bioeng. 2006, 93, 169-180. (21) Torisawa, Y.; Kaya, T.; Takii, Y.; Oyamatsu, D.; Nishizawa, M.; Matsue, T. Anal. Chem. 2003, 75, 2154-2158.

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of a collagen gel matrix in a silicon microstructure. The proliferation rate of these cells conformed to that in vivo;23 hence, it could be concluded that they maintained their in vivo-like nature. The cellular viability was evaluated by monitoring the cellular respiratory activity by using scanning electrochemical microscopy (SECM). This technique uses a microelectrode as a scanning probe to monitor the local distribution of electroactive species.24 The SECM method enables the monitoring of various cellular functions, e.g., enzymatic activity,25 the detoxication function,26 and nitric oxide release.27 Electrochemical detection systems offer the following advantages: the ability to miniaturize, integration of multiple sensor electrodes on a chip, and detection in real time.28-31 In this study, we have investigated the in situ cellular signal transduction of 3-D cultured cells by applying electrochemical methods. These cells were genetically engineered to produce SEAP and immobilized in an array of microholes on a chip. Cellular SEAP expression triggered by the exposure to tumor necrosis factor R (TNFR) was monitored using two kinds of electrochemical detection systems. First, the cellular SEAP expression activity was continuously monitored by employing an SECM-based assay and compared with the cellular viability. Further, a detection electrode array was integrated into the cellular chip and applied to monitor cellular SEAP expression in parallel. Our study aims to realize a high-throughput screening system for cellular gene expression. EXPERIMENTAL SECTION Chemicals. p-Aminophenylphosphate monosodium salt (PAPP; LKT Lab Inc.), p-aminophenol (PAP; Wako Pure Chemical Industries), TNFR (Wako Pure Chemical Industries), bovine serum albumin (BSA; Wako Pure Chemical Industries), poly(dimethylsiloxane) (PDMS; KE-106, Shin-etsu Chem. Co. Ind. Ltd.), tetramethylammonium hydroxide (Tokyo Kasei Kogyo Co., Ltd.), and all other chemicals were used as received. Cell Culture and Transfection. The human breast cancer cell line (MCF-7) used in this study was donated by the Cell Resource Center for Biomedical Research (Tohoku University). The cells were cultured in RPMI-1640 medium (Gibco Invitrogen, Tokyo, Japan) containing 10% fetal bovine serum (FBS; Gibco), 50 µg mL-1 penicillin (Gibco), and 50 µg mL-1 streptomycin (Gibco) at 37 °C in a humidified atmosphere containing 5% CO2. (22) Torisawa, Y.; Shiku, H.; Yasukawa, T.; Nishizawa, M.; Matsue, T. Biomaterials 2005, 26, 2165-2172. (23) Torisawa, Y.; Shiku, H.; Kasai, S.; Nishizawa, M.; Matsue, T. Int. J. Cancer 2004, 109, 302-308. (24) Bard, A. J.; Fan, F.-R. F.; Mirkin, M. V. In Electroanalytical Chemistry; Bard, A. J., Ed.; Marcel-Dekker: New York, 1994; Vol. 18. (25) Feng, W.; Rotenberg, S. A.; Mirkin, M. V. Anal. Chem. 2003, 75, 41484154. (26) Mauzeroll, J.; Bard, A. J.; Owhadian, O.; Monks, T. J. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 17582-17587. (27) Isik, S.; Etienne, M.; Oni, J.; Blochl, A.; Reiter, S.; Schuhmann, W. Anal. Chem. 2004, 76, 6389-6394. (28) Sullivan, M. G.; Utomo, H.; Fagan, P. J.; Ward, M. D. Anal. Chem. 1999, 71, 4369-4375. (29) Held, M.; Schuhmann, W.; Jahreis, K.; Schmidt, H.-L. Biosens. Bioelectron. 2002, 17, 1089-1094. (30) Andreescu, S.; Sadik, O. A.; McGee, D. W. Anal. Chem. 2004, 76, 23212330. (31) Popovtzer, R.; Neufeld, T.; Biran, D.; Ron, E. Z.; Rishpon, J.; ShachamDiamand, Y. Nano Lett. 2005, 5, 1023-1027.

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MCF-7 cells were transfected with the pSEAP2-Control, pSEAP2Basic, or pNFκB-SEAP vector (BD Sciences). The pSEAP2 series is an integrated set of plasmids that differ only with regard to the presence or absence of the simian virus 40 (SV40) promoter or enhancer sequence. The pSEAP2-Control vector contains the SEAP structural gene that is under the transcriptional control of the SV40 promoter and enhancer, whereas the pSEAP2-Basic vector contains the SEAP gene without a promoter and enhancer.17 On the other hand, the pNFκB-SEAP vector encodes SEAP under the control of four copies of the κB enhancer element. By employing these vector sets, various cellular signal transductions can be detected by the corresponding enhancer elements. The cells were seeded in a 35-mm dish (Falcon) at a density of 5 × 105 cells in 2 mL of RPMI-1640 medium containing 10% FBS without antibiotics. A day after the cultivation, transfection was performed by the addition of 500 µL of Opti-MEM I medium (Gibco) containing 4 µg of plasmid DNA and 10 µL of LipofectAMINE 2000 (Invitrogen); this was followed by incubation for 5 h. Subsequently, the transfection medium was changed to a pure culture medium, and the cells were incubated at 37 °C overnight. Chip Preparation. The substrate for the cell panel array consisted of 5 × 5 panels of pyramid-like cavities micromachined on a silicon wafer.21 A (100) silicon wafer (230 µm thick) was diced into 20 × 20 mm2 squares and then oxidized in a wet oxygen atmosphere at 1000 °C for 10 h to produce an oxide layer. Quadrilateral windows (450 × 450 µm2) were photolithographically patterned onto the oxide layer to define the larger openings of the microhole. The pyramidal cavities were etched using anisotropic etching in 25% (CH3)4NOH at 80 °C. The small openings of the pyramidal hole were 150 × 150 µm2 in size; they were used for the SECM-based measurements, as illustrated in Figure 1a. The volume of each pyramidal microhole was ∼20 nL. The transfected cells were centrifuged and suspended in an 8:1:1 mixture of type I collagen (Cellmatrix Type I-A, Nitta Gelatin), MEM medium, and a reconstituted buffer solution. The final cell density in the collagen-cell mixture was 1 × 107 cells mL-1. A few microliters of the collagen-cell mixture was dropped on the larger opening microholes of the silicon substrate. The bilateral side of the silicon substrate was then sandwiched by PDMS sheets adsorbed with BSA to facilitate the detachment from the collagen gel.32 After a 20-min incubation at 37 °C for gelation of the collagen-cell mixture, the PDMS sheets were peeled off in a PBS solution. Approximately 200 cells were immobilized in the 20-nL microholes. The cell chip thus obtained was incubated overnight before the experiments. SECM-Based Assay. Cellular SEAP expression was evaluated by using PAPP as an electrochemical substrate. The product, PAP, can be oxidized as shown in Figure 1. The SECM measurements were conducted in a buffered isotonic solution comprising 10 mM HEPES, 150 mM NaCl, 4.2 mM KCl, and 11.2 mM glucose. The PAP oxidation current was recorded using a platinum electrode in a HEPES-based saline solution (pH 9.5) containing 5 mM PAPP. The potential was maintained at 0.3 V versus Ag/AgCl to specifically evaluate the PAP oxidation current (see Figure 1b). Except for PAP, no other electroactive species exists at 0.3 V. The PAPP oxidation current was observed to increase at a more (32) Tang, M. D.; Golden, A. P.; Tien, J. J. Am. Chem. Soc. 2003, 125, 1298812989.

Figure 1. (a) Principle of the SECM-based SEAP assay using a cell chip. A collagen-cell mixture was embedded into pyramid-like cavities on the silicon substrate. A Pt microelectrode was set at a potential of 0.3 V vs Ag/AgCl and scanned 30 µm above the silicon substrate at a scan rate of 19 µm s-1. (b) The cyclic voltammograms of 5 mM PAPP and 5 mM PAP taken with a Pt microelectrode (radius, 5 µm) in a HEPESbased saline solution (pH 9.5). The scan rate was 20 mV s-1.

Figure 2. (a) Schematic illustration of the electrode-integrated cell culture device. A Pt electrode was located around the smaller opening of the microhole. (b-d) Micrograph of the device. (c) Magnification image of the silicon microhole integrated with the Pt electrode. (d) Two PDMS wells were placed on both sides of the silicon substrate. The cells were embedded within the microhole and cultured on the larger opening side. For the SEAP measurement, the device was laid on a glass slide with the larger opening side facing downward. (e) The cyclic voltammogram of 4 mM ferrocyanide measured with the surrounding Pt electrode on the chip. The scan rate was 20 mV s-1.

positive potential than +0.4 V. The Pt microelectrode for the SECM study was fabricated to have a radius of ∼5.0 µm, as determined from the voltammograms of ferrocyanide.33 A motordriven XYZ stage (Suruga Seiki, K701-20.R) was located on the microscope stage for microelectrode tip scanning. The cellular chip was immersed in a culture dish with a 2-mL measurement solution with the larger opening side at the bottom. The microelectrode tip was held at a distance of 30 µm above the silicon substrate and then scanned over the collagen-cell panels at a scan rate of 19 µm s-1. The time required to obtain an image of 1 mm × 1 mm2 at a spatial resolution of 10 µm was within 100 min. The respiratory activity of the cells that were embedded within the cell panels was evaluated with SECM, as previously reported.21 The oxygen reduction current was recorded at -0.5 V versus Ag/ (33) Wightman, R. M. Anal. Chem. 1981, 53, 1125A-1134A.

AgCl in the HEPES-based saline solution (pH 7.2). The tip was scanned at a distance of 30 µm above the chip surface to obtain a single 1-mm-width scan within 60 s. The cellular respiratory activity was assessed by the decrease in oxygen reduction current, which was normalized to the base current (typically, 1.5 nA) corresponding to the oxygen concentration in the measuring solution. The decrease in the oxygen reduction current indicated the corresponding change in respiratory activity. Data were obtained for more than three panels for every experiment, and the values represent the mean ( SD of at least 10 experiments that were conducted on each date. SEAP Assay Using the Electrode-Integrated Cell Culture Device. We fabricated a cell culture device by integrating a Pt electrode around the silicon microhole, as shown in Figure 2. A silicon substrate fabricated with 2 × 4 panels of microholes was oxidized to insulate it from the bulk substrate. After the patterning Analytical Chemistry, Vol. 78, No. 22, November 15, 2006

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Figure 3. (a) Optical micrograph of a cell panel. MCF-7 cells were embedded in a collagen gel matrix on a silicon microstructure. (b, c) SECM images of the SEAP activity for the cell-panel-integrated cells transfected with pSEAP2-Control (b) or pSEAP2-Basic (c) in a HEPES-based saline solution containing 5 mM PAPP. The tip was scanned over an area of 1 mm × 1 mm at a scan rate of 19 µm s-1.

of photoresist, a Pt layer (thickness, 200 nm) with a Ti adhesion layer was deposited onto the substrate by using a sputterdeposition apparatus (L-3325, Anelva). After the liftoff of the photoresist, a photodefinable polyimide was patterned to define the shape of the electrode. The 25-µm-wide Pt electrode was located around the smaller opening of the microhole. For the cell culture and the measurement, two PDMS wells were placed on both sides of the device.22 A mixture of PDMS and a curing agent was poured into 2-mm interspaces between fluoridized glass slides, and these were heat-cured at 100 °C for 1 h. The PDMS well was fabricated using a CO2 laser engraving system (Universal Laser System Inc.). The 30-W CO2 laser beam was scanned over the PDMS layer to pierce a well. The PDMS well on the larger opening side was for the cell culture medium and that on the smaller opening side was for the solution that was used for the electrochemical measurement. The electrochemical measurement was conducted as follows: after washing out the culture medium on the larger opening side, the device was laid on a glass slide with the larger opening side facing downward, as shown in Figure 2d. For the electrochemical solution, a 500-µL HEPES-based saline solution was added on the smaller opening side of the PDMS well, and Ag/AgCl reference and platinum counter electrodes were placed in the well. Ten minutes after applying 0.3 V versus Ag/AgCl at the working electrodes integrated on the silicon substrate, a 500-µL HEPESbased solution containing 10 mM PAPP was added. The current measurement was regulated with a multipotentiostat (Hokuto Denko, HA1010mM4). Chemiluminescence SEAP Assay. The SEAP activity in the culture medium was evaluated by the chemiluminescence method by using a Great EscAPe SEAP detection kit (BD Biosciences). The transfected MCF-7 cells were centrifuged and suspended in a collagen solution, and 30-µL droplets were subsequently placed in sterile 96-well plates at a density of 1 × 104 cells well-1. After gelation by 30-min incubation at 37 °C, 200 µL of the culture medium was overlaid and incubated overnight. The conditioned medium (15 µL) was mixed with 45 µL of a dilution buffer and then incubated at 65 °C for 30 min to eliminate endogenous 7628 Analytical Chemistry, Vol. 78, No. 22, November 15, 2006

alkaline phosphatase activity. After cooling to room temperature, the samples were mixed with 60 µL of an assay buffer containing L-homoarginine for 5 min, and this mixture was then added to 60 µL of a chemiluminescence solution containing 1.25 mM disodium 3-(4-methoxyspiro(1,2-dioxetane-3,2-(5-chloro)tricyclo[3.3.1.1.3,7]decan)-4-yl)phenyl phosphate (CSPD, BD Biosciences) substrate. After incubation in the dark for 30 min, chemiluminescence signals were detected using a luminometer (Luminometer 20, Promega). The assays were performed in triplicate. Succinic Dehydrogenase Inhibition Assay. Cellular succinic dehydrogenase activity was assessed by a 2-(2-methoxy-4-nitrophenyl)-3- (4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-8) colorimetric assay according to the protocol of a cell counting kit (Dojindo). MCF-7 cells were seeded into sterile 96-well plates at a density of 1 × 104 cells well-1 in 100 µL of a culture medium. After overnight cultivation, 10 µL of a medium containing TNFR was added at a final concentration of 100 ng mL-1, and the plates were incubated for a further 24 or 48 h. A 10-µL aliquot of the WST-8 assay solution was then added to each well, and the plates were incubated for 4 h. The optical density was measured at 450 nm using a microplate reader (model 680, Bio-Rad Labs, Inc.). The cellular viability was evaluated as a percentage of the control. The assays were performed in triplicate. RESULTS AND DISCUSSION SECM-Based SEAP Assay. We report here, for the first time, an evaluation of the SEAP expression of 3-D cultured cells on a chip that was monitored by SECM as shown in Figure 1a. The cells were embedded in a collagen gel matrix and trapped in the silicon microstructure. SECM measurement was carried out in the HEPES-based saline solution of pH 9.5. It was observed that almost all the cells were alive in the measurement solution for at least 2 h, using the SDI and the fluorescent viability assays.22 The SEAP activity of two kinds of cellssthose transfected with a pSEAP2-Control or a pSEAP2-Basic vectorswas imaged by SECM. The cells that were transfected with pSEAP2-Control continuously secreted SEAP, whereas those transfected with pSEAP2-Basic did not secrete SEAP at all.17 Therefore, the cell

panels in which the collagen-cell mixture containing the pSEAP2Control and pSEAP2-Basic vectors were embedded act as a positive and a negative control, respectively. Figure 3 shows the optical image (a) and SECM images of the cell panel transfected with pSEAP2-Control (b) and pSEAP2-Basic (c). The SECM image for the positive control cells shows a bright spot with a high oxidation current; the location of the spot coincides with that of the cell panel. On the contrary, no change in the current was observed in the SECM image for the negative control cells. The increase in the oxidation current around the cell panel is a consequence of the cellular SEAP expression. Although it took ∼100 min to image the cell panel, the oxidation current profile was almost symmetrical and in a steady state. The background current was also found to be almost constant. Thus, the SEAP enzyme continuously diffuses from the cell panel, indicating that the oxidation current profile reflects the SEAP concentration profile. The SECM-based assay can monitor cellular SEAP expression in situ, and there is no need for any additional pretreatment, such as heat treatment or the addition of an inhibitor, in the assay procedure in order to eliminate endogenous alkaline phosphatase activity. The SEAP activity was also evaluated by the conventional chemiluminescence assay to confirm the SEAP expression of the 3-D cells in the collagen gel. Figure 4a shows the time course of the SEAP expression of the positive and negative control cells (104 cells well-1). The chemiluminescence due to SEAP expression was detectable 12 h after cultivation. The SEAP expression of the positive control cells showed a linear increase for 24 h, proving that the rate of cellular SEAP secretion was constant. On the other hand, the expression of the negative control cells was not observed at all. Therefore, it can be stated that the SEAP reporter works properly in the collagen gel-embedded culture system and that the sensitivity and selectivity of the SECM-based assay is comparable to the conventional chemiluminescence assay. Additionally, equivalent responses are observed in the case of both the positive and negative control cells. Characterization of Cellular Signal Transduction by the Chemiluminescence Assay. Cellular SEAP secretion via nuclear factor κB (NFκB) activation that was induced by exposure to TNFR was characterized as a model system to study cellular signal transduction. The cells were transfected with pNFκB-SEAP to produce SEAP under the control of the NFκB enhancer elements. When the cells were stimulated by TNFR, NFκB was activated and translocated to the nucleus, resulting in the induction of SEAP expression. Therefore, NFκB signal transduction can be evaluated by detecting SEAP activity. Figure 4b shows the SEAP activity assessed by chemiluminescence detection. Before the stimulation, the SEAP activity was at a negligible level. SEAP expression was induced 3 h after the stimulation with 100 ng mL-1 TNFR; subsequently, the expression continuously increased for 48 h. When the cells were cultured in the absence of TNFR, the SEAP activity showed a slight increase, indicating that NFκB was moderately activated under the typical culture condition. At this point, it is noteworthy that the chemiluminescence intensity reflects the amount of accumulated SEAP in the culture medium because of the sampling method in the assay protocol. Figure 4c shows the amount of increased SEAP expression in the corresponding time period. When the cells were

Figure 4. SEAP activity assayed by chemiluminescence detection. (a) Time course of SEAP expression in cells transfected with pSEAP2Control or pSEAP2-Basic. The measurements were started 12 h after adding the culture medium into the 96-well plate. (b) Time course of SEAP induction following exposure to TNFR. Cells were transfected with pNFκB-SEAP and cultured in the absence or presence of 100 ng mL-1 TNFR. (c) The increase in SEAP expression for the corresponding time period after the exposure to TNFR.

stimulated by TNFR, SEAP expression drastically increased until 12 h and then decreased, whereas in the absence of stimulation, the expression was very low and almost constant. The above results indicate that SEAP induction occurs 3 h after the TNFR stimulation and reaches a maximum at 12 h. Although the chemiluminescence assay can monitor the time course of SEAP activity by the medium-sampling method, it is difficult to monitor cellular SEAP expression in situ or to design a higher throughput system based on chemiluminescence measurements. Analytical Chemistry, Vol. 78, No. 22, November 15, 2006

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Table 1. Cellular Viability (% of Control) for TNFr

SECM measurement SDI assay

Figure 5. Electrochemical characterization of SEAP induction and respiratory activity following exposure to TNFR. (a) Current response in one-line scan over the center of a cell panel before (a-1) and 3 (a-2), 6 (a-3), 12 (a-4), 24 (a-5), and 48 h (a-6) after exposure to a medium containing 100 ng mL-1 ΤΝFR. (b) Time course of the increase in the PAP oxidation current. Cells were transfected with pNFκB-SEAP or pSEAP2-Basic and cultured in a medium containing 100 ng mL-1 TNFR. (c) Time course of cellular respiratory activity evaluated by SECM. The cell chip was incubated in the absence or presence of 100 ng mL-1 TNFR. The respiratory activity was normalized to that obtained before the addition of TNFR (0 h).

Cellular Signal Transduction Monitored by SECM. Figure 5a shows the single-scan SECM responses captured on the cell panels after exposure of the culture medium to TNFR. The microelectrode was laterally scanned over the center of the cell panel for 1 mm. Figure 5b shows the time course of SECM responses following exposure to TNFR on the cellular chip transfected with pNFκB-SEAP or pSEAP2-Basic. The Y-axis represents the peak response in the single-scan measurements near the cell panel. Before the stimulation, the oxidation current increased slightly near the cell panel, proving that NFκB was 7630 Analytical Chemistry, Vol. 78, No. 22, November 15, 2006

24 h

48 h

85 ( 9.4 90 ( 10

59 ( 11 60 ( 11

moderately activated by the culture medium. SEAP expression drastically increased up to 12 h after stimulation and then decreased. The results obtained by the SECM-based assay showed a tendency similar to those obtained by the chemiluminescence assay. The SECM measurement can continuously monitor SEAP activity in situ for several days because of its noninvasive feature. The SEAP expression of cells transfected with pSEAP2-Basic was scarcely induced. Therefore, the current increase was attributed to the cellular secretion of SEAP resulting from NFκB activation. The SECM-based assay for SEAP expression using cellular microarrays enables the evaluation of SEAP expression in multiple cellular microarrays and, finally, elucidates cellular signal transduction. The respiratory activity can also be monitored by SECM to monitor the local oxygen reduction current at the microelectrode probe. Figure 5c shows the time course of the respiratory activity after exposure to 100 ng mL-1 TNFR. The respiratory activity was normalized to that before the addition of TNFR. It increased after the addition of TNFR. Since cells consume excess oxygen34,35 to produce reactive oxygen species (ROS) when they are stimulated by TNFR, the increase in the respiratory activity is attributed to cellular oxygen consumption along with the production of ROS. Therefore, this result indicates that the cellular oxygen consumption increased. It appeared that the cellular production of ROS increased until 6 h and continued for 18 h. Furthermore, the respiratory activity 24 h after the stimulation was lower than that without stimulation. This result implies that the cellular viability decreased because of the cytotoxic effect of TNFR. Table 1 shows the cellular viability obtained by the SECM-based assay and the SDI assay, a popular method for conducting cellular viability tests. The cytotoxic effect of TNFR increased with an increase in the duration of exposure. The results obtained by the SECM-based assay are in good agreement with those obtained by the SDI assay, suggesting that the SECM-based assay does not affect the cellular viability even if several measurements are carried out. Therefore, the SECM-based assay can simultaneously detect cellular signal transduction and respiratory activity based on the cellular viability or oxygen consumption. Evaluation of SEAP Activity Using the Electrode-Integrated Cell Culture Device. To allow practical applications, we have also evaluated SEAP activity by using a cellular chip with an integrated electrode array. Figure 2 shows the schematic view (a) and optical micrograph of the cell culture device (b-d). The Pt microelectrode (width, 25 µm; inner circumference, 600 µm; outer circumference, 800 µm) was located around the smaller opening of the silicon microhole and insulated using a polyimide. Two PDMS wells were placed on both sides of the silicon substrate as reservoirs. Figure 2e shows the cyclic voltammogram (34) Mouithys-Mickalad, A.; Deby-Dupont, G.; Nys, M.; Lamy, M.; Deby, C. Biochem. Biophys. Res. Commun. 2001, 287, 781-788. (35) Garg, A. K.; Aggarwal, B. B. Mol. Immunol. 2002, 39, 509-517.

Figure 6. Electrochemical detection of SEAP activity using the cell culture device. Two kinds of cellssthose transfected with pSEAP2-Control and pSEAP2-Basic (a) or pNFκB-SEAP and pSEAP2-Basic (b)swere embedded on the left and right sides on the same chip, respectively. At the point indicated by the arrow, PAPP (final concentration, 5 mM) was added to the HEPES-based saline solution. (b) The measurement was carried out 12 h after exposure to a medium containing 100 ng mL-1 TNFR.

of 4 mM ferrocyanide measured with the surrounding Pt electrode on the chip. The steady-state current (150 nA) is close to that (120 nA) theoretically expected for a ring electrode with a 600µm inner and 800-µm outer circumference, and lower than that (270 nA) expected for a band electrode that is 25 µm wide and 700 µm long. This result suggests that the polyimide resist on the electrode effectively limits the active area of the working electrode. SEAP activity was evaluated by using an electrode-integrated cell culture device. Two kinds of transfected cells were immobilized on the left and right sides on the same chip for comparative characterization of their SEAP activity. Figure 6a shows the current responses of the positive and negative control cells. The oxidation current observed for the positive control cells increased for 20 min after the addition of PAPP (final concentration 5 mM) and remained almost constant at a high response level. Alternatively, the current response for the negative control cells scarcely increased. The increase in the background current (∼1 nA) was observed in both the positive and negative control cells immediately after the addition of PAPP. This background response is attributed to the oxidation of PAPP. The response observed for the positive control cells represents the expression of cellular SEAP. Figure 6b shows the current responses of the pNFκB-SEAPtransfected and negative control cells that were simultaneously monitored with two panels in the device. Electrochemical measurement was carried out 12 h after the exposure of the device to a medium containing 100 ng mL-1 TNFR. The current responses showed a tendency similar to that observed in Figure 6a; the response of the pNFkB-SEAP-transfected cells increased for 20 min, whereas such a response was not observed in the negative control cells. It should be noted that the response obtained in the case of the pNFκB-SEAP-transfected cells was larger than that obtained in the positive control cells. This tendency is in good agreement with that observed in the SECM-based measurements. (36) Nagamine, K.; Onodera, S.; Torisawa, Y.; Yasukawa, T.; Shiku, H.; Matsue, T. Anal. Chem. 2005, 77, 4278-4281.

These results demonstrate that the electrochemical assay with the integrated device enables the evaluation of SEAP activities in multiple cellular microarrays. The electrode-integrated cellular microdevice will be developed further for its application to highthroughput monitoring of the cellular expression of various genes in real time. CONCLUSIONS In this study, we demonstrated electrochemical reporterexpression assays to monitor cellular signal transduction by using SECM-based and electrode-integrated cell culture devices. The SECM-based assay allows in situ monitoring of cellular SEAP expression with high reliability. Furthermore, simultaneous measurements of cellular signal transduction and respiratory activity were accomplished by SECM. The electrochemical reporter assay was also performed by using a device in which an electrode array was integrated with the cellular chip. The electrode-integrated, cell-based microarray system can be applied to a high-throughout screening system. Our group previously reported on-chip transformation by using an array of silicon microholes predeposited with different plasmid DNAs.36 The development of the SEAP assay on the reverse transfection microarrays would be a useful tool for monitoring cellular gene expression. ACKNOWLEDGMENT This work was partly supported by a Grant-in-Aid for Scientific Research on Priority Areas (445) from the Ministry of Education, Culture, Sports, Science and Technology of the Government of Japan, and by a Grant-in-Aid for Scientific Research (17710112) from the Ministry of Education, Science and Culture, Japan. Y.T. acknowledges the support obtained from a research fellowship of the Japan Society for the Promotion of Science. Received for review April 18, 2006. Accepted September 7, 2006. AC060737S

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