Single-Cell Chemical Lysis Method for Analyses of Intracellular

Nov 4, 2008 - Chem. , 2008, 80 (23), pp 9141–9149 ... at the single-cell level: detection of proteins by antibody conjugated microbeads and measurem...
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Anal. Chem. 2008, 80, 9141–9149

Single-Cell Chemical Lysis Method for Analyses of Intracellular Molecules Using an Array of Picoliter-Scale Microwells Yasuhiro Sasuga,†,‡ Tomoyuki Iwasawa,† Kayoko Terada,†,‡ Yoshihiro Oe,†,§ Hiroyuki Sorimachi,† Osamu Ohara,*,|,⊥ and Yoshie Harada†,‡,§,# The Tokyo Metropolitan Institute of Medical Science, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan, Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), Honcho, Kawaguchi, Saitama 332-0012, Japan, Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Building FSB-401, 5-1-5, Kashiwanoha, Kashiwa, Chiba 277-8562, Japan, Department of Human Genome Research, Kazusa DNA Research Institute, 2-6-7 Kazusa-kamatari, Kisarazu, Chiba 292-0818, Japan, Research Center for Allergy and Immunology, RIKEN Yokohama Institute, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan, and Institute for Integrated Cell-Material Sciences (iCeMS), Kyoto University, Yoshida-konoecho 69, Sakyo-ku, Kyoto 6068501, Japan Analyzing the intracellular contents and enzymatic activities of single cells is important for studying the physiological and pathological activities at the cellular level. For this purpose, we developed a simple single-cell lysis method by using a dense array of microwells of 10-30pL volume fabricated by poly(dimethylsiloxane) (PDMS) and a commercially available cell lysis reagent. To demonstrate the performance of this single-cell lysis method, we carried out two different assays at the single-cell level: detection of proteins by antibody conjugated microbeads and measurement of protease activity by fluorescent substrates. The results indicated that this method readily enabled us to monitor protein levels and enzymatic activities in a single cell. Because this method required only an array of PDMS microwells and a fluorescence microscope, the simplicity of this platform opens a way to explore the biochemical characteristics of single cells even by those who are not familiar with microfluidic technology. A cell is the fundamental unit of life, and all functions in multicellular organisms are ultimately attributed to those of the cell. Although most conventional biochemical assays are performed using a considerable number of cells to determine their quantitative biomolecular profiles, such bulk assays only provide their averaged values in the analyzed ensemble and thus often overlook important information regarding their fluctuations among individual cells.1,2 Because the differences among individual cells in the same ensemble are highly critical in some cases such as * To whom correspondence should be addressed. Phone: +81-438-52-3913. Fax: +81-438-52-3914. E-mail: [email protected]. † The Tokyo Metropolitan Institute of Medical Science. ‡ CREST, JST. § The University of Tokyo. | Kazusa DNA Research Institute. ⊥ RIKEN Yokohama Institute. # Kyoto University. (1) Ferrells, J. E.; Machleder, E. M. Science 1998, 280, 895–898. (2) Levsky, J. M.; Singer, R. H. Trends Cell Biol. 2003, 13, 4–6. 10.1021/ac8016423 CCC: $40.75  2008 American Chemical Society Published on Web 11/05/2008

cell differentiation, quantitative measurement of biomolecular profiles at the single-cell level is a matter of concern in the biological community. Should it be possible to determine the biochemical parameters of each of multiple single cells in parallel, we would be able to describe the behavior of individual cells in an ensemble for obtaining deeper insights into cell-cell signaling, genetic heterogeneity, and heterotypic biological activities.3 Furthermore, if single-cell biochemical analysis becomes feasible, we will be able to save much time, labor, and cost because we no longer have to purify a cell ensemble to homogeneity. To develop a single-cell analysis in quantitative biology, various methods have been actively explored.4-8 However, these newly emerging methods have not been fully applied in the biological sciences yet. One of the reasons for this is the fact that these methods are too sophisticated and integrated to be used without appropriate investment of time, money, and labor. Thus, there is a strong need to simplify and make these methods more biologistfriendly for realization of quantitative biology at the single-cell level. We believed that the difficulty lies in the fact that quantitative analysis of intracellular biological contents at the single-cell level must seamlessly integrate multiple steps: cell isolation/trapping, cell lysis, and quantitative assays of biochemical contents in the lysate. As for multiplexed single-cell analyses, most methods reported thus far take advantage of highly sophisticated and automated instruments for integration of these multiple steps on a single platform, e.g., single-cell capture followed by chemical lysis in a closed volume of 50 pL recently reported in a micro(3) Rubin, M. A. Science 2002, 296, 1329–1330. (4) Levsky, J. M.; Shenoy, S. M.; Pezo, R. C.; Singer, R. H. Science 2002, 297, 836–840. (5) Hong, J. W.; Studer, V.; Hang, G.; Anderson, W. F.; Quake, S. R. Nat. Biotechnol. 2004, 22, 435–439. (6) Kurimoto, K.; Yabuta, Y.; Ohinata, Y.; Ono, Y.; Uno, K. D.; Yamada, R. G.; Ueda, H. R.; Saitou, M. Nucleic Acids Res. 2006, 34, e42. (7) Huang, B.; Wu, H.; Bhaya, D.; Grossman, A.; Granier, S.; Kobilka, B. K.; Zare1, R. N. Science 2007, 315, 81–84. (8) Newman, J. S.; Ghaemmaghami, S.; Ihmels, J.; Breslow, D. K.; Noble, M.; DeRisi, J. L.; Weissman, J. S. Nature 2006, 441, 840–846.

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fabricated device.9 However, the complicated flow path and process of the microfabricated device in that report could be a serious drawback for extending the application to single-cell biochemistry. Although the idea of a highly integrated laboratoryon-a-chip device for culturing, sorting, trapping, and lysing cells and analyzing their contents is overwhelmingly attractive,10-12 it will be some time before such a laboratory-on-a-chip device becomes easy and robust to use for biologists. Thus, as an alternative approach, we intended to develop a total single-cell analysis system in an extremely simplified format. In this regard, the most serious concerns were the procedure for lysing single cells in a closed space and quantitative detection of molecules in a single cell after cell lysis. Importantly, the sensitivity of the detection method must be high enough to enable us to monitor the quantity of biomolecules in a single cell. Therefore, to achieve quantitative single-cell analysis, we must at least address these two concerns. In this context, we here describe the establishment of a simple microwell-based single-cell lysis method for the determination of intracellular proteins or enzymatic activities at the single-cell level. This approach includes (1) cell trapping in an array of picoliterscale microwells, (2) chemical lysis in the closed microwell, and (3) fluorescent detection of immunosignals or enzymatic activities in the microwell. As we and other groups13-16 have already reported some basic elements of this method, in this study, we have focused our analyses particularly on evaluating the performance of the chemical single-cell lysis approach. Although physical trapping of a single cell in a microwell has already been reported by other groups,13-15 chemical cell lysis in the closed trapped microwell was first examined in this study. Although there are a wide variety of single-cell lysis methods in the literature,17 an important advantage of this approach is that this method enables us to chemically lyse a large number of single cells in an array of picoliter-scale microwells separately and simultaneously without any sophisticated microfluidic technology. Because this cell lysis method requires only a microwell array, the entire setup is extremely simple. Furthermore, to achieve quantitative biochemical assays at the single-cell level, we combined this singlecell lysis method with highly sensitive fluorescence microscopic techniques because fluorescence microscopy is one of the methods most familiar to biologists. Capillary electrophoresis with laser-induced fluorescent detection has been widely applied for single-cell analysis.18,19 We applied the microbead-based immunofluorescent detection for the quantification of intracellular proteins and fluorogenic substrate-based assays for enzymatic (9) Irimia, D.; Tompkins, R. G.; Toner, M. Anal. Chem. 2004, 76, 6137–6143. (10) Navratil, M.; Whiting, C.E.; Arriaga, E. A Sci STKE 2007, pe29. (11) Wu, H.; Wheeler, A.; Zare, R. N. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 12809–12813. (12) Price, A. K.; Culbertson, C. T. Anal. Chem. 2007, 79, 2614–2621. (13) Rettig, J. R.; Folch, A. Anal. Chem. 2005, 77, 5628–5634. (14) Revzin, A.; Sekine, K.; Sin, A.; Tompkins, R. G.; Toner, M. Lab Chip 2005, 5, 30–37. (15) Ostuni, E.; Chen, C. S.; Ingber, D. E.; Whitesides, G. M. Langmuir 2001, 17, 2828–2834. (16) Sasuga, Y.; Tani, T.; Hayashi, M.; Yamakawa, H.; Ohara, O.; Harada, Y. Genome Res. 2006, 16, 132–139. (17) Brown, R. B.; Audet, J. J. R. Soc. Interface 2008;published online. (18) Huang, W.-H.; Zhong-Li, F. A.; Cheng, J.-K. J. Chromatogr., B 2008, 866, 104–122. (19) Munce, N. R.; Li, J.; Herman, P. R.; Lilge, L. Anal. Chem. 2004, 76, 4983– 4989.

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activity mainly because of its simplicity and scalability. Our results indicate that the combination of this single-cell lysis method with highly sensitive fluorescence microscopy would pave the way to quantitatively analyze biomolecules or enzymatic activities at the single-cell level in reality. EXPERIMENTAL SECTION Reagents. Polyclonal antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), Sigma-Aldrich (St. Louis, MO), Medical & Biological Laboratories Co., Ltd. (Nagoya, Japan), or GE Healthcare UK Ltd. (Buckinghamshire, England). The monoclonal anti-FLAG epitope tag was supplied by SigmaAldrich. For antibody immobilization, carboxylated polystyrene microbeads and dimethyl pimelimidate · 2HCl (DMP) were purchased from Bangs Laboratories Inc. (Fishers, IN) and Pierce (Rockford, IL), respectively. For microbead array fabrication, sulfosuccinimidyl 6-(4′-azido-2′-nitrophenylamino) hexanoate (sulfoSANPAH) was supplied by Pierce. Fluorogenic substrates and peptide aldehyde were purchased from the following manufacturers: succinyl-L-leucyl-L-leucyl-L-valyl-L-tyrosine 4-methylcoumaryl7-amide (Suc-LLVY-MCA), acetyl-L-aspartyl-L-glutamyl-L-valyl-Laspartic acid R-(4-methylcoumaryl-7-amide) (Ac-DEVD-MCA), 7-amino-4-methylcoumarin (AMC), and carbobenzoxy-L-leucinylL-leucinal (Z-Leu-Leu-H) from Peptide Institute (Osaka, Japan) and bis(N-CBZ-L-aspartyl-L-glutamyl-L-valyl-L-asparticacidamide)rhodamine 110 (Z-DEVD-R110) and rhodamine 110 from Calbiochem (San Diego, CA). The calpain inhibitor calpastatin was supplied by TaKaRa Bio (Shiga, Japan). Cell Culture. Rat pheochromocytoma PC12 cells were cultured in Dulbecco’s modified Eagle medium (DMEM; with 1000 mg/mL glucose and sodium bicarbonate, Sigma-Aldrich) supplemented with 4 mM L-glutamine, 5% horse serum (Invitrogen Corp, Carlsbad, CA), and 10% Fetal Clone III (Hyclone Laboratories Inc., Logan, UT) at 37 °C in the presence of 5% CO2. The cells were subcultured every 3-4 days. In the case of induction of apoptosis, serum was completely removed by washing the cells three times with PBS (8000 mg/L NaCl, 200 mg/L KCl, 1150 mg/L Na2HPO4, 200 mg/L KH2PO4), and the cells were then cultured in a serumfree medium for several hours. Cell Staining. Nuclear staining was performed by adding 20 µg/mL Hoechst 33342 (Molecular Probes, Eugene, OR) in medium for 20 min at 37 °C in the presence of 5% CO2. Cell surface staining was performed as follows. The cells were washed and collected in PBS and treated with 1 µM amino-reactive Cy3-dye (GE Healthcare UK Ltd.) for 5 min at room temperature. After two washes each with PBS and medium, the stained cells were seeded on a poly(dimethylsiloxane) (PDMS) sheet. For differential immunofluorescent staining of PC12 cells transfected with expression plasmids for BAP-HA and FLAG-GST, the PC12 cells were fixed with 4% paraformaldehyde in PBS and allowed to react with rabbit anti-GST antibody (G7781; Sigma-Aldrich) and mouse antiHA antibody (JM-3996-13; Medical & Biological Laboratories Co., Ltd.). After reaction with (a) antibodies, followed by (b) extensive washing with PBS containing 0.1% Tween 20, BAP-HA and FLAGGST were differentially detected by the Cy5-labeled antirabbit IgG antibody and Cy3-labeled antimouse IgG antibody (PA5004 and PA43002, respectively; GE Healthcare UK Ltd.). Plasmids and Transfection. cDNAs encoding the FLAGtagged GST and FLAG and HA-tagged bacterial alkaline phos-

phatase (BAP) genes were prepared by PCR and cloned into pTriEX-3 hygro (Novagen, San Diego, CA). PCR amplification was performed with a high-fidelity DNA polymerase, Pyrobest DNA polymerase (TaKaRa Bio). Transfections were performed using 4 µL of Lipofectamine 2000 reagent (Invitrogen Corp.) per 35mm dish according to the manufacturer’s instructions. The cells were transfected with 1.5 µg each of the above plasmids and pEGFP-N2 (TaKaRa Bio) in several combinations. The transfected cells were maintained for 24-48 h at 37 °C under 5% CO2. For optimizing the concentration of the cell lysis solution, we used cloned PC12 cells stably transformed with pEGFP-N2. PDMS Microwell Array Fabrication. Microwell array fabrications were performed by Fluidware Technologies Inc. (Tokyo, Japan). The master molds were fabricated by using silicon wafers and the chromic masked photolithography method. PDMS was cast over the master mold to create a complementary microwell array in PDMS. The overall shape of the PDMS sheet was designed to be 20 mm × 20 mm with a thickness of 1 mm. A microwell array was constructed on the PDMS sheet in a central area of 10 mm × 10 mm. Microwell arrays with three different well sizes were fabricated (areas: 40 µm × 40 µm, 30 µm × 30 µm, and 20 µm × 20 µm with a constant depth of 20 µm). The wellto-well interval was 30 µm. Each PDMS sheet thus contained a microwell array consisting of ∼20 000 microwells. Seeding of Cells in Microwells. Microwells tend to trap air bubbles due to the hydrophobicity of PDMS. To avoid air bubbles, a PDMS sheet was placed under vacuum for several minutes before the cell culture medium was dispensed onto the sheet. A suspension of cells was diluted to a concentration of ∼2 × 107 cells/mL in an appropriate medium and a 100-µL aliquot was pipetted onto the surface of the PDMS sheet immersed in the same medium (3 mL) in a 35-mm dish. The cells were allowed to settle for ∼12 h at 37 °C in the presence of 5% CO2. The surface of the PDMS sheet was then scraped with a coverslip to remove excess of the cell suspension and further washed with PBS for removing the cells that had not adhered to the microwells. The percentage of microwells occupied by the cells and the average number of cells per well were determined by counting them in randomly determined viewing fields by using a 10× objective lens. Single-Cell Lysis in Microwells. An array of microwells treated as described above was placed open-side down on a coverslip (24 × 32 mm, Matsunami Glass Ind., Ltd., Osaka, Japan). In this case, the PDMS sheet and the coverslip were separated by two double-faced adhesive tapes (Nichiban Co., Ltd., Tokyo, Japan) and the space between them served as a flow cell for solution change in microwells. The flow cell volume was ∼20 µL. The following procedures were conducted on the platform of a fluorescence microscope. To lyse the cells, CelLytic-M (SigmaAldrich) appropriately diluted with PBS was poured into the flow cell, and the PDMS sheet was quickly pressed with an acryl bar to close the microwells. These procedures were completed within 20 s. The entire assembly was incubated at room temperature for 30-120 s. The appearance of the cell lysis process was monitored under a microscope with a 40× objective lens in real time. Microbead Preparation. Protein A (Pierce) was fixed to carboxylated polystyrene microbeads with an average diameter of 0.95 µm (PC03N, Bangs Laboratories Inc., Fishers, IN) by a chemical cross-linking reaction as described in a previous

method.16 Antibody-conjugated microbeads were prepared as follows: Anti-GST or anti-HA antibodies (sc-459 or sc-805, respectively; Santa Cruz Biotechnology, Inc.) were first trapped on protein A-conjugated microbeads and then cross-linked with protein A on the beads by using 6.5 mg/mL DMP in 0.2 M triethanolamine (pH 8.2). Microbead Array Fabrication. Sulfo-SANPAH solution (0.05 mg/mL in 0.1 M sodium phosphate buffer, pH 7.2) was poured into the flow cell generated between two amino-silanated coverslips by using two adhesive tapes, as described in the previous section and incubated for 20 min. After incubation with a blocking solution [0.5 mg/mL dephosphorylated R-casein (Sigma-Aldrich) in PBS buffer] for 15 min, the upper coverslip was removed and an array of cell-charged microwells was placed open well-side down on the sulfo-SANPAH-treated coverslip. The antibodyconjugated microbeads were placed in the flow cell, and a single microbead was captured by an optical trap. After moving under the target cell, the microbead was placed on the glass surface and illuminated with UV (300-360 nm) light for 1-5 s. The unbound microbeads were washed away by using PBS after fixation. The single-cell lysis step was then carried out as described above. Immunodetection of Specific Proteins. After lysing a cell with an array of antibody-conjugated microbeads, the entire assembly was incubated at room temperature for 20 min. When the pressure was released, the PDMS sheet reverted to its initial open state, and the flow cell was flushed with a secondary antibody binding buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% Tween 20, 0.5 mg/mL dephosphorylated R-casein, pH 7.4). After incubation for 10 min, a Cy3-labeled anti-FLAG antibody (Sigma-Aldrich, 10 nM in secondary antibody binding buffer) was injected into the flow cell and the resultant mixture was incubated at room temperature for 20 min. Subsequently, the secondary antibody was washed, and the Cy3 fluorescence on the microbeads was observed with a 100× objective lens. Just before observation, an oxygen scavenger system was added to prevent fluorescence bleaching.20 Single-Cell Enzyme Assay. The following buffers were used for measuring the activities of calpain and caspase-calpain assay buffer: 100 mM Tris-HCl, 20 mM 2-mercaptoethanol, 0.1% CHAPS, 2 mM AEBSF, 2 µM pepstatin A, 200 nM Aprotinin, 200 µM TLCK, 2 µM phosphoramidon, 500 nM MG-132, 200 µM bestatin, and 45%CelLytic-M;pH7.5.Caspaseassaybuffer:100mMHEPES-KOH, 10% sucrose, 0.1% CHAPS, 10 mM DTT, 1 mM PMSF, 2 µg/mL chymostatin, 2 µg/mL leupeptin, 2 µg/mL antipain, 2 µg/mL pepstatin A, 45% CelLytic-M; pH 7.5. Suc-LLVY-MCA was used for the calpain assay, and Ac-DEVD-MCA or Z-DEVD-R110 was applied for the caspase assay. Each fluorogenic substrate was diluted with the respective assay buffer at a concentration of 100 µM. The single-cell lysis step was carried out as described above by using a reaction mixture of interest. Fluorescence was observed using a 40× objective lens under a fluorescence microscope (described below), and the images were recorded every 30 s. Molar concentrations of the produced free dye were calculated from the fluorescence intensity of free AMC and rhodamine110. All reactions were observed at room temperature. (20) Harada, Y.; Sakurada, K.; Aoki, T.; Thomas, D. D.; Yanagida, T. J. Mol. Biol. 1990, 216, 49–68.

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Microscope. An inverted microscope (IX-70, Olympus Co., Tokyo, Japan) was modified to fit the microbead array platform, as described in a previous method.16 Nd:YAG laser (model IRCL1000-1064, 500 mW, CrystaLaser, Longley Lane, NV) and UV light from a mercury lamp were used for optical manipulation and photoactivation, respectively. These laser and UV lights were illuminated through a 40× objective lens (UApo/340 40×, NA ) 1.35, Olympus Co.). A 532-nm laser (model Compass 315M-100 SL, 100 mW, Coherent Inc., Santa Clara, CA), a 488-nm laser (model Sapphire 488-20, 20 mW, Coherent Inc.), or a mercury lamp was used with appropriate filter sets for fluorescence excitation through a 40× or 10× objective lens (PlanApo 100×, NA ) 1.40, Olympus Co.). Excitation light intensity and exposure time were controlled by neutral density filters and a mechanical shutter. Bright-field and fluorescence images were captured by an EBCCD camera with an image intensifier or SIT camera (Hamamatsu Photonics, Hamamatsu, Japan). For imaging of cell lysis, a 488nm laser and EB-CCD camera were used with a 40× objective lens. For microbead array analysis, a 532-nm laser and EB-CCD camera were applied with a 100× objective lens. For imaging of enzymatic activities, a mercury lamp and an SIT camera were used with a 40× objective lens. The acquired images were recorded using a digital video cassette recorder (DSR-11, Sony Co., Tokyo, Japan). Quantitative measurements of fluorescence intensity were performed with Meta Imaging Software (Molecular Devices, Sunnyvale, CA). RESULTS AND DISCUSSION Principle of the Cell Lysis Method. To generate a simple cell lysis system, we adopted a microfabricated device composed of PDMS, a silicon elastomer that is transparent to visible light and adhesive to smooth surfaces. Overall procedures of our microwell-based, single-cell lysis method are schematically shown in Figure 1a. A suspension of cells was deposited onto an array of microwells, and the cells were allowed to settle into the microwells randomly as described in previous reports by other groups.13,14 After removing the excess cells and medium by scraping and aspiration, a flow cell was constructed between a bottom coverslip and a top cell-trapped PDMS sheet, which were separated by two double-faced adhesive tapes [Figure 1a-(3)]. Since PDMS is flexible, the PDMS sheet could be easily deformed by pressing. After the cell lysis solution diffuses into a microwell from the flow cell, the PDMS sheet is rapidly pressed against the bottom coverslip to close each microwell and thereby allow gradual cell lysis. PDMS Microwell Design and Single-Cell Capture. For realizing the procedures described above, an array of microwells was fabricated by a combination of photolithography and replica molding of PDMS. The arrays comprised micrometer-sized cavities of three different sizes (areas: 40 µm × 40 µm, 30 µm × 30 µm, and 20 µm × 20 µm) with a constant depth of 20 µm and a well-to-well spacing of 30 µm. The array was designed to fit within the 24 mm × 32 mm coverslip, with a thickness of 1 mm (Figure 1b). The solution in the microwells was replaced by passing it in a flow cell constructed between the coverslip and the PDMS sheet. The flow cell volume was ∼20 µL. By pressing the PDMS sheet 9144

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Figure 1. Single-cell capture and lysis. (a) Schematic drawing of the single-cell lysis procedures: (1) A suspension of cells is deposited onto an array of microwells fabricated in a PDMS sheet; (2) after cells are allowed to settle in the wells, the excess cells are removed by aspiration; (3) the PDMS sheet harboring the cells is inverted to form a flow cell with a bottom coverslip; (4) a cell lysis reagent is applied to the flow cell; (5) the PDMS sheet is pressed against the bottom coverslip to close the open side of the microwell; (6) the cells are gradually lysed in the closed microwells. (b) An overview of the PDMS sheet (scale bar, 10 mm) and a microscopic image of the microwells (scale bar, 100 µm). (c) A microscopic image of a PC12 cell-trapped microwell array (microwell size, 40 µm × 40 µm × 20 µm; scale bar, 50 µm).

with an acryl bar, we succeeded in repeated closing/opening rounds and in replacing the solution in the microwells through flushing 3-fold flow cell volumes with the new solution (see Supporting Information Figure S-1). To carry out cell lysis in microwells, we first seeded the cells on the surface of a PDMS sheet at an appropriate dilution. The number of cells deposited per well depended on the density of the deposited cell suspension and on the size of the microwells. For a PDMS sheet containing 20 000 microwells (40 µm × 40 µm × 20 µm), we found that 3 mL of a cell suspension, containing ∼2 × 106 cells (2 × 107 cells/mL × 0.1 mL), seeded for 12 h, yielded one to three cells in ∼40% of the wells (Figure 1c and see Supporting Information Figure S-2a). Cells trapped in the microwells and submerged in a large culture medium divided normally for more than 4 days in culture (see Supporting Information Figure S-2b). Thus, the array of microwells composed of PDMS enabled us to trap and grow a single cell in each well in accordance with a previous report for mouse hybridoma cells.21 It must be noted that the deposited PC12 cells adhered onto the PDMS sheet even when inverted because the cells were nonspecifically adhered to PDMS. Single-Cell Lysis in PDMS Microwells. We selected CelLytic-M (Sigma-Aldrich), a commercially available mammalian cell lysis reagent, to chemically lyse cells in microwells. This reagent is an appropriate detergent solution designed for efficient wholecell protein extraction from cultured mammalian cells, and the resultant lysates can be used for various downstream applications (e.g., reporter gene assays, immunoblotting/immunoprecipitation, electrophoretic mobility shift assays, phosphatase assays, and kinase assays) without removing the reagent. These well(21) Love, J. C.; Ronan, J. H.; Grotenbreg, G. M.; Veen, A. G.; Ploegh, H. L. Nat. Biotechnol. 2006, 24, 703–707.

Figure 2. Lysis of a single EGFP-producing cell. (a) Microscopic images showing the lysing of an EGFP-expressing PC12 cell. The left panel shows the bright-field image and the EGFP fluorescence merged image of a trapped cell in a microwell before the addition of CelLytic-M. The right panel shows the diffusion of EGFP throughout the microwell in ∼1 s. Time was measured from the time point of cell lysis. The white arrowhead points to a trapped single cell. Scale bar, 25 µm. (b) Observation of an EGFP-producing cell before and after lysis at low magnification. Subsequent monitoring after lysis of an EGFP-expressing PC12 cell for over 20 min. Scale bar, 100 µm. Cell lysis was performed using 45% CelLytic-M.

characterized features of this cell lysis reagent from a biochemical viewpoint were highly crucial because they could guarantee the performance of this reagent at the single-cell biochemistry level. However, in order to optimize the protocols of our single-cell lysis method with this reagent, we had to first set up the most suitable experimental lysis conditions by monitoring a lysis process of EGFP-expressing PC12 cell in a PDMS microwell. We first optimized the concentration of the cell lysis solution by using EGFP-producing PC12 cells. As only the cytoplasm of EGFP-producing PC12 cells fluoresces brightly before cell lysis while EGFP fluorescence is diffused throughout a microwell after cell lysis, we could easily follow the cell lysis processes under a fluorescence microscope (Figure 2). When the concentration of the lysis solution is extremely high, cell lysis takes place very rapidly to entrap the cell lysate in the microwell. On the contrary, when the concentration is extremely low, cell lysis barely occurs. Because the optimized concentration of the lysis solution could vary from cell to cell, we considered that the preliminary experiments should be performed with a cell of interest in advance. Figure 3 shows the results of the optimization of the concentration of the lysis solution by using EGFP-expressing PC12 cells. In the case of a high concentration, lysis efficiency was satisfactory, but the cell holding rate was very low since most entrapped cells peeled off from the microwell at the time of injection. We found that 40-50% of the CelLytic-M solution would be best for PC12 cells because it induced almost complete lysis of cells while the cell holding rate was still acceptable (Figure 2). Thus, we used ∼2-fold diluted CelLytic-M reagent in the following experiments. In the presence of 45% CelLytic-M solution, the cell was lysed within 2 min, and the cytoplasmic EGFP diffused readily in a microwell (Figure 2a and see Supporting Information video 1). We found that the cell lysate did not leak into the next microwell even after incubation for over 20 min, using the protocols shown in Figure 1a (Figure 2b). We further investigated the possibility of lysing the nucleus and the cell membrane by this method because this is a critical concern in the biochemical analyses of

Figure 3. Optimizing the concentration of CelLytic-M for single-cell lysis in microwells. Cell lysis was identified by EGFP diffusion of a single PC12 cell throughout the microwell under various CelLytic-M concentrations. The lysis efficiency was obtained by dividing the number of lysed cells by the total number of EGFP-producing cells (closed circle, the left vertical axis). The cell holding rate (open triangle, right vertical axis) was calculated by dividing the number of microwells with fluorescent cells (or lysate) by the total number of microwells in a microscopic view, which provided us measurement of the retaining efficiency of the cell lysate in a microwell under the conditions where 40-50% of the microwells were occupied by EGFPproducing cells. Measurements were performed in triplicate for each experimental setting.

nuclear and membrane proteins. As a model, we monitored the lysis process of an EGFP-expressing PC12 cell in which the nuclear DNA or cytoplasmic cell membrane proteins were stained using fluorescence staining, as described in the Experimental Section. When a single cell with fluorescently stained nuclear DNA was lysed in a microwell under the conditions described above, the nuclear shape remained fluorescently visible, whereas the cytoplasmic EGFP became readily diffused throughout the microwell (see Supporting Information Figure S-3a). On the other hand, we succeeded in lysing the cell membrane by this method because the fluorescence of Cy3-labeled cell surface proteins diffused throughout a microwell as EGFP did (see Supporting Information Figure S-3b). The microwell-based, cell lysis method thus enabled us to successfully lyse the cytoplasmic membrane and hold the cell lysate in a subnanoliter-scale microwell, whereas the nuclei could not be completely solubilized. Since EGFP with nuclear localization signal was observed to be released from the nuclei by this method, we considered that the nuclear envelope might be considerably perturbed. Incomplete lysis of the nuclei of PC12 cells probably resulted from lowering the concentration of the CelLytic-M reagent to 50%, which was much lower than that recommended by the supplier. Taken together, we considered this method to be the most suited one for the biochemical analyses of cytoplasmic proteins and cytoplasmic membrane proteins at the single-cell level. While the use of microfluidic devices is one of the methods of choice to perform single-cell lysis in nanoliter flow streams,7,11,12,22 it requires a complicated flow path and process for analysis and quantification of the molecule of interest. In contrast, our method is extremely simple. Cell lysis was completed in merely two steps: reagent injection and pressing the PDMS sheet onto the coverslip. (22) He, M.; Edgar, J. S.; Jeffries, G. D. M.; Lorenz, R. M.; Shelby, J. P.; Chiu, D. T. Anal. Chem. 2005, 77, 1539–1544.

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More importantly, the dilution of cellular analytes could be kept to a minimum by this method. Application of Microbead Array for the Detection of a Specific Protein in a Single Cell. To evaluate the applicability of this new single-cell lysis method for the specific detection of intracellular proteins at the single-cell level, we first examined the detection of recombinant proteins produced in a single cell by combining this method with an antibody-microbead array method previously developed by us.16 Because the dimension of this microbead array is extremely smaller than that of conventional microarrays, this technique makes it possible to quantify specific proteins even in a subnanoliter volume, which is close to the volume of a single animal cell. For example, 9 microbeads (3 × 3 pattern) were easily arrayed on a coverslip, which served as the bottom of a microwell holding a single cell (see Supporting Information Figure S-4), implying that it is possible, in principle, to combine this microbead-based array method with the PDMS microwell-based single-cell lysis method. Next, we tried to detect specific proteins in a single animal cell by using the microwell-assisted, cell lysis method coupled with the antibody-immobilized microbead array method. For this purpose, we measured the production levels of recombinant FLAGtagged GST (FLAG-GST) produced by a single PC12 cell. For the specific detection of FLAG-GST, we conducted an antibody sandwich assay by using capture antibody (anti GST antibody)immobilized microbeads and Cy3-labeled detection antibody (anti FLAG antibody). Then, the FLAG-GST-expressing PC12 cells were seeded onto PDMS microwells (40 µm × 40 µm × 20 µm), and the PDMS sheet was assembled into a flow cell as described in the Experimental Section. For the quantification of the produced FLAG-GST and for checking its specificity, anti-GST and anti-HA tag antibody-coated microbeads were arrayed side by side under the target cell to be included in the microwell. In this case, the anti-HA immobilized bead served as a negative control. The cell was lysed as described in the Experimental Section. An incubation time of 20 min was selected because it was considered to be sufficient to attain equilibrium in the antibody-antigen binding reaction in the microwell, as judged from our previous report.16 After incubation, the PDMS sheet was opened and the lysate was replaced with a buffer solution for the antibody sandwich assay by supplying it through the gap between the PDMS sheet and the coverslip. To detect the captured FLAG-GST onto probe microbeads, the Cy3-labeled anti-FLAG antibody was applied into the flow cell. We observed Cy3-fluorescence only on anti-GST antibody-coated beads; no fluorescence was observed on the antiHA tag antibody-coated beads (Figure 4a). The fluorescence intensity of the antibody-coated microbeads increased with the concentration of FLAG-GST as expected and started to saturate at ∼2 nM FLAG-GST (see Supporting Information Figure S-5). Although the dynamic detection range of the FLAG-GST concentration could vary depending on various conditions (the excitation intensity, sensitivity of the CCD camera, reaction time, number of antibodies captured on microbeads), it was between 0.2 and 10 nM under the conditions presented in Supporting Information Figure S-5. If we estimate the concentration of FLAG-GST from the fluorescence intensities on the microbeads by using the calibration curve in Supporting Information Figure S-5, the 9146

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Figure 4. Detection of intracellular proteins of a single cell. (a) Microscopic images showing the detection of FLAG-GST produced by a single PC12 cell. Left and middle panels show the bright-field image, while the right panel is a Cy3-fluorescence image of the same microwell. Cy3-fluorescence corresponded to the specific detection signal of FLAG-GST. Black and white arrowheads point to the trapped single cell and immobilized microbeads, respectively. Scale bar, 10 µm. (b) Analyses of production of BAP-HA and coproduction of FLAGGST and BAP-HA: left panel, bright-field image; right panel, Cy3fluorescence image. Cy3-fluorescence also corresponded to the specific detection signals of FLAG-GST and BAP-HA. Scale bar, 10 µm.

concentration of FLAG-GST in Figure 4a was estimated to be ∼4 nM. Similar experiments were performed using a different PC12 cell, which expressed FLAG- and HA-tagged BAP (BAP-HA). In this case, while the Cy3 fluorescence was detected on anti-HA antibody-coated microbeads as expected, no fluorescence was observed on anti-GST antibody-coated beads (upper panel in Figure 4b). In addition, when FLAG-GST and BAP-HA were coexpressed in PC12 cells, fluorescence was detected on both antiGST antibody-coated and anti-HA tag antibody-coated beads (lower panel in Figure 4b). These results indicated that the detection system was specific as defined by antibody specificity. The BAP-HA/FLAG-GST ratio in the cell population was reproducible among the experimental runs judging from the results of immunoblot analyses of the cell lysate (data not shown). However, interestingly enough, the protein production profiles of FLAG-GST and BAP-HA obtained by the microbead system varied widely from cell to cell although they coexpressed both proteins (Figure 5). This observation was further verified by the differential fluorescent immunostaining of FLAG-GST and BAPHA in the transfected cells (Supporting Information Figure S-6); the ratios of the immunofluorescent signal of BAP-HA against that of FLAG-GST varied as widely as those shown in Figure 5. Thus, these results suggested that this method enabled us to monitor cell-to-cell variations of intracellular protein profiles, which could not be achieved by conventional biochemical analysis using many cells as an ensemble. Because we analyzed the cell lysate in the same microwell immediately after cell lysis, loss of cellular contents was very unlikely. In addition, we could not detect any nonspecific capture of antigens onto the PDMS microwell, coverslip, or microbead

Figure 5. Analysis of protein production levels of FLAG-GST and BAP-HA coproduced by PC12 cells. (a) Fluorescence images after detection of the Cy3-labeled secondary antibody. Fifteen single PC12 cells cotransfected with expression plasmids for FLAG-GST and BAPHA were analyzed. (b) Ratios of fluorescence intensities (FI) observed for FLAG-GST and BAP-HA. The fluorescence ratios were calculated by dividing the FI of BAP-HA/FI of FLAG-GST.

under the blocking conditions employed. Therefore, the sensitivity of this microbead-based analysis platform is considered to be primarily determined by the affinities of the antibodies used for the sandwich assay. For example, if we tried to detect the target molecules using an antibody array in a subnanoliter-volume well, the detection limit of the target molecules would be several thousand molecules per cell since the detection limit of the molecules by using Cy3-based sandwich detection is as low as 0.2 nM, as demonstrated in our previous and current studies (Supporting Information Figure S-5).16 This detection limit is considered to be low enough to monitor the protein profiles of major proteins in a single cell. To further improve the sensitivity, several groups have attempted to prepare biochip applications in which the probe molecules are oriented in the appropriate direction.23-25 In fact, the platform for the oriented buildup of immunoglobulin on a gold surface exhibited enhancement in the functional immunoglobulin density for efficient antigen captures.24 If such a protein-oriented method becomes applicable for probe microbead preparation, the sensitivity of this microbead-based analysis platform will be greatly improved because our fluorescent detection system shows single-fluorophore sensitivity by itself. This is also expected to considerably improve the dynamic range of the analyte concentration. An arraying step of microbeads inside the microwell might become a drawback of this microbead-based analysis upon increase of the number of single cells or antigens to be assayed, because we have manually arrayed microbeads by (23) Abad, A. M.; Mertens, S. F. L.; Pita, M.; Fernndez, V. M.; Schiffrin, D. J. J. Am. Chem. Soc. 2005, 127, 5689–5694. (24) Ha, T. H.; Jung, S. O.; Lee, J. M.; Lee, K. Y.; Lee, Y.; Park, J. S.; Chung, B. H. Anal. Chem. 2007, 79, 546–556. (25) Shmanai, V. V.; Nikolayeva, T. A.; Vinokurova, L. G.; Litoshka, A. A. BMC Biotechnol. 2001, 1, 4–8.

optical trapping in this study. Thus, another microbead-arraying method as well as automation of the optical trapping is now being explored for future development of this analysis platform. Application of Fluorogenic Substrate for the Detection of a Specific Enzyme Activity in a Single Cell. In addition to the quantity of proteins, the enzymatic activity in a cell extract is a matter of general concern. To address enzymatic activity at the single-cell level, we took advantage of the enzyme-specific substrate containing the fluorogenic leaving group.26-29 As a model, we first measured the intracellular Ca2+-dependent protease (calpain)30,31 activity at the single-cell level by combining it with the microwell-based chemical cell lysis method and fluorogenic measurement of enzymatic activity. PC12 cells were seeded and lysed in the presence of a calpain substrate (Suc-LLVY-MCA) as described in the Experimental Section. After incubation for 10 min, we monitored the fluorescence of the generated products under a fluorescence microscope. When a single cell was trapped in a microwell, the fluorescence in the microwell was detected due to the release of the free fluorophore from the substrate, whereas fluorescence was barely observed in the empty microwell (Figure 6a). Real-time monitoring of the enzymatic activities was also performed under several conditions. The fluorescence intensity in the microwell increased over time in the presence of Ca2+, whereas a weak signal was observed in the presence of EDTA, probably because of incomplete calcium chelation. Moreover, when a calpain inhibitor such as Z-Leu-Leu-H27 or calpastatin32,33 was added, the calpain activities of single cells were completely inhibited even in the presence of Ca2+ as expected (Figure 6b and c). These results indicated that we could successfully monitor the protease activity specific to calpain by using these protocols. We further tested another enzyme assay. Caspases (cysteine aspartate-specific protease) are known to be key players in activating apoptosis in vertebrate cells,34,35 and their enzymatic activities are widely used as a measure of apoptosis induced by various stimuli. PC12 cells, without exposure to nerve growth factors, are dependent on serum; the withdrawal of serum is known to initiate apoptosis.36,37 Caspase 3 activity was thus measured after serum withdrawal by using bulk cell lysates in a preliminary experiment. Robust caspase activity was detected after (26) Leytus, S. P.; Melhado, L. L.; Mangel, W. F. Biochem. J. 1983, 209, 299– 307. (27) Tsubuki, S.; Saito, Y.; Tomioka, M.; Ito, H.; Kawashima, S. J. Biochem. 1996, 119, 572–576. (28) Tompa, P.; Buzder-Lantos, P.; Tantos, A.; Farkas, A.; Szila´gyi, A.; Ba´noczi, Z.; Hudecz, F.; Friedrich, P. J. Biol. Chem. 2004, 279, 20775–20785. (29) Hug, H.; Los, M.; Hirt, W.; Debatin, K. Biochemistry 1999, 38, 13906– 13911. (30) Goll, D. E.; Thompson, V. F.; Li, H.; Wei, W.; Cong, J. Physiol. Rev. 2003, 83, 731–801. (31) Suzuki, K.; Hata, S.; Kawabata, Y.; Sorimachi, H. Diabetes 2004, 53, S12– S18. (32) Asada, K.; Ishino, Y.; Shimada, M.; Shimojo, T.; Endo, M.; Kimizuka, F.; Kato, I.; Maki, M.; Hatanaka, M.; Murachi, T. J. Enzyme Inhib. 1989, 3, 49–56. (33) Takano, J.; Tomioka, M.; Tsubuki, S.; Higuchi, M.; Iwata, N.; Itohara, S.; Maki, M.; Saido, T. C. J. Biol. Chem. 2005, 280, 16175–16184. (34) Vaux, D. L.; Strasser, A. Proc. Natl. Acad. Sci. U. S. A. 1996, 93, 2239– 2244. (35) Thornberry, N. A.; Lazebnik, Y. Science 1998, 281, 1312–1316. (36) Mesner, P. W.; Winters, T. R.; Green, S. H. J. Cell Biol. 1992, 119, 1669– 1680. (37) Vaghefi, H.; Hughes, A. L.; Neet, K. E. J. Biol. Chem. 2004, 279, 15604– 15614.

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Figure 6. Detection of single-cell enzymatic activities. (a) Fluorescence images of the calpain activity of a single PC12 cell. Fluorescence images 10 min after lysis. The white arrowhead represents a trapped single cell. Scale bar, 10 µm. (b) Continuous monitoring of calpain activities of a single cell under several conditions. Images were collected at 60-s intervals. Ca2+, 10 mM CaCl2; EDTA, 25 mM EDTA; Ca2++ZLLH, 10 mM CaCl2 and 1 mM Z-Leu-Leu-H; and Ca2++CLST, 10 mM CaCl2 and 140 µM calpastatin in the cell lysis reagent. Scale bar, 10 µm. (c) Time courses of molar concentrations of the free dye produced. (d) Detection of caspase 3 activities of single cells by using a caspase 3 substrate, Z-DEVD-R110. Two types of PC12 cells were analyzed: one, inducing apoptosis by withdrawing the serum 3 h beforehand, and the other, without withdrawing the serum. The histogram of fluorescence intensities at 10 min after lysis in the microwells. The lines represent a Gaussian fit.

3 h of serum withdrawal as expected (see Supporting Information Figure S-7). Then, caspase 3 activity after serum withdrawal for 3 h in single PC12 cells was measured using a fluorogenic substrate for caspase 3 (Ac-DEVD-MCA or Z-DEVD-R110) by this microwell-based chemical cell lysis method. Caspase 3 activation was observed upon serum withdrawal at the single-cell level (Figure 6d and see Supporting Information Figure S-8). Interestingly enough, the data indicated that caspase 3 activity is highly variable at the single-cell level before and after serum withdrawal. Because no two cells have an identical cell cycle and response to environmental stimulation, this cell-to-cell variation of caspase 3 activity may be due to the stochastic nature of caspase 3 activation or due to the heterogeneity of the cell status. As described above, we successfully detected native intracellular enzymatic activities at the single-cell level. Although a similar microwell array format for measuring enzymatic activity of single molecules using a fluorogenic substrate was reported by Rondelez et al.,38 ours is, to our knowledge, the first report on the measurement of enzymatic activities in a single-cell lysate by using a microwell array. Although in situ measurements of enzyme activity might be possible if a cell membrane-permeable substrate is available,29,39 this is not always the case. More importantly, the chemical single-cell lysis method developed in this study potentially enables us to measure the quantity of enzyme and its enzymatic activity in parallel. In addition, since multicolor imaging is possible in these analyses, we could measure multiple different kinds of information from the same single cells. In fact, we could (38) Rondelez, Y.; Tresset, G.; Tabata, K. V.; Arata, H.; Fujita, H.; Takeuchi, S.; Noji, H. Nat. Biotechnol. 2005, 23, 361–365. (39) Boonacker, E.; Elferink, S.; Bardai, A.; Fleischer, B.; Van Noorden, C. J. F. J. Histochem. Cytochem. 2003, 51, 959–968.

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successfully carry out the annexin V fluorescent assay40 in parallel with caspase enzymatic measurements (data not shown). CONCLUSIONS In this study, we have developed a novel platform for singlecell lysis by a combination of an array of micrometer-sized wells with a commercially available cell lysis reagent. Using the flow cell constructed with a PDMS microwell array, we succeeded in trapping and lysing single cells in each of subnanoliter-scaled wells, thus confirming that the resulting single-cell lysates could be successfully used for measuring the quantity of intracellular proteins and the activities of endogenous enzymes. Our system had several unique features compared to conventional microfluidic single-cell lysis systems: (1) simple protocols consisting of merely two steps for lysis, (2) minimized dilution of cell lysate during lysis, and (3) parallel processing of an ensemble of single cells. Together with highly sensitive fluorescent detection techniques, our approach has opened a way to analyze the heterogeneity of a cell population at the single-cell level from a biochemical viewpoint. ACKNOWLEDGMENT This work was supported in part by a research grant from Core Research for Evolutional Science and Technology (CREST) of Japan Science and Technology Agency (JST), by a Grant-in-Aid for Scientific Research by the Ministry of Education, Culture, Sports, Science and Technology of Japan (to Y.H.), by the Human Frontier Science Program (to Y.H.), and by research grants from Kazusa DNA Research Institute and RIKEN Research Center for (40) Koopman, G.; Reutelingsperger, C. P. M.; Kuijten, G. A. M.; Keehnen, R. M. J.; Pals, S. T.; Oers, M. H. J. Blood 1994, 84, 1415–1420.

Allergy and Immunology (to O.O.). Y.H. also acknowledges financial support from Toray Science Foundation. SUPPORTING INFORMATION AVAILABLE Details of the buffer exchange; Figure S-2, distribution of microwell occupancies; Figure S-3, cell lysis; Figure S-4, microbead array fabrication; Figure S-5, antibody sandwich detection; Figure S-6, cell-to-cell variation data obtained by differential fluorescent

immunostaining; Figures S-7 and S-8, measurements of Caspase 3 activity in single PC12 cells. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review April 6, 2008. Accepted October 13, 2008. AC8016423

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