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Detection of Cytochrome c in a Single Cell Using an Optical Nanobiosensor Joon Myong Song, Paul M. Kasili, Guy D. Griffin, and Tuan Vo-Dinh*
Advanced Biomedical Science and Technology Group, Life Sciences Division, Oak Ridge National Laboratory, P.O. Box 2008, Oak Ridge, Tennessee 37831-6101
In this work, the intracellular measurement of cytochrome c using an optical nanobiosensor is demonstrated. The nanobiosensor is a unique fiberoptics-based tool which allows the minimally invasive analysis of intracellular components. Cytochrome c is a very important protein to the process which produces cellular energy. In addition, cytochrome c is well-known as the protein involved in apoptosis, or programmed cell death. δ-Aminolevulinic acid (5-ALA) was used to induce apoptosis in MCF-7 human breast carcinoma cells. 5-ALA, a photodynamic therapy (PDT) drug in cells was activated by a HeNe laser beam. After the PDT photoactivation, the release of cytochrome c from the mitochondria to the cytoplasm in a MCF-7 cell was monitored by the optical nanobiosensor inserted inside the single cell and followed by an enzymelinked immunosorbent assay (ELISA) outside the cell. The combination of the nanobiosensor with the ELISA immunoassay improved the detection sensitivity of the nanobiosensor due to enzymatic amplification. Our results lead to the investigation of an apoptotic pathway at the single cell level. Apoptosis is programmed cell death, which is very different from necrosis, cell death from catastrophic outside forces.1,2 It is an important process for a diseased cell to die because it does not become cells with viral infections, damaging cells around it, but rather shrinks and collapses inward causing little damage to surrounding cells. This is a normal part of tissue growth and occurs throughout our bodies. Apoptosis is also induced when a cell detects irreparable DNA damage. In this case, P53, a proapoptotic gene present in all cells, has put the cell into the most extreme form which leads to the death pathway.3,4 Photodynamic therapy (PDT) is an important medical treatment technology that uses lasers to activate light-sensitive photosensitizers to treat cancer and other diseases in a nonsurgical, minimally invasive way.5-9 The unique property of PDT drugs is to preferentially accumulate in neoplastic tissues. After PDT drugs * Corresponding author. Phone: (865) 574-6249. E-mail:
[email protected]. (1) Littlewood, T. D.; Bennett, M. R. Curr. Opin. Lipidol. 2003, 14, 469-475. (2) Kwon, Y. W.; Masutani, H.; Nakamura, H.; Ishii, Y.; Yodoi, J. Biol. Chem. 2003, 384, 991-996. (3) Robinson, M.; Jiang, P.; Cui, J.; Li, J.; Wang, Y.; Swaroop, M.; Madore, S.; Lawrence, T. S.; Sun, Y. Cancer Biol. Ther. 2003, 2, 406-415. (4) Manfredi, J. J. Mol. Cell 2003, 11, 552-554. (5) Kamuhabwa, A. A.; Roskams, T.; D’Hallewin, M. A.; Baert, L.; Van Poppel, H.; De Witte, P. A. Int. J. Cancer 2003, 107, 460-467. (6) Dolmans, D. E.; Fukumura, D.; Jain, R. K. Nat. Rev. Cancer 2003, 3, 380387. 10.1021/ac0352878 CCC: $27.50 Published on Web 04/03/2004
© 2004 American Chemical Society
are injected into humans, the excitation light appropriate for a given drug and disease is delivered through the illuminating device and irradiates the tissues. The PDT drug is then excited to its singlet state, which is extremely active. The excited PDT drug interacts with molecular oxygen to produce highly reactive and cytotoxic “singlet” oxygen. As a result, the direct tumorocidal activity and microvascular damage destroys tumor cells. It has been reported that tumor cell death by PDT is related to the induction of apoptosis, and mitochondrial localized photosensitizers are able to induce apoptosis very rapidly.10,11 Cytochrome c is a protein that is important to the process of creating cellular energy, the main function of mitochondria. When mitochondria are damaged by PDT, cytochrome c is released into the cytoplasm of the cell. The release of cytochrome c is part of the cascade of cellular events that lead to apoptosis.12 This indicates that measurement of cytochrome c in the cytoplasm can be used as evidence that apoptosis is occurring. This can also lead to greater understanding of certain diseases on a cellular level. Gao, et al. reported that breast cancer cells under the influence of a protein called hepatocyte growth factor/scatter factor (HGF/SF), which are then subjected to treatment with chemotherapeutic drugs, do not release cytochrome c.13 These cells do not undergo apoptosis that chemotherapeutic drugs should induce. Cytochrome c release from mitochondria, as indicative of apoptosis, is also observed in certain neurological conditions. It has been discovered that the pathway of cell death triggered by mitochondrial release of cytochrome c is a likely cause of the damage and death of the motor neurons, the nerve cells that control muscle function, seen in the disease amyotrophic lateral sclerosis.14 This suggests that elucidating the many steps of the cascade of mitochondrial-induced cell death might lead to the development of medications that can block some steps. This may offer a treatment for the largely untreatable disease. (7) Haddad, R.; Kaplan, O.; Greenberg, R.; Siegal, A.; Skornick, Y.; Kashtan, H. Int. J. Surg. Invest. 2000, 2, 171-178. (8) Yang, V. X.; Muller, P. J.; Herman, P.; Wilson, B. C. Lasers Surg. Med. 2003, 32, 224-32. (9) Chou, T. M.; Woodburn, K. W.; Cheong, W. F.; Lacy, S. A.; Sudhir, K.; Adelman, D. C.; Wahr, D. Catheter Cardiovasc. Interv. 2002, 57, 387-394. (10) Moor, A. C. E. J. Photochem. Photobiol. 2000, 57, 1-13. (11) Tempestini, A.; Schiavone, N.; Papucci, L.; Witort, E.; Lapucci, A.;Cutri, M.; Donnini, M.; Capaccioli, S. Eur. J. Ophthalmol. 2003 (Suppl 3), S11-18. (12) Lam, M.; Oleinick, N. L.; Nieminen, A. L. J. Biol. Chem. 2001, 276, 4737947386. (13) Gao, M.; Fan, S.; Goldberg, I. D.; Laterra, J.; Kitsis, R. N.; Rosen, E. M. J. Biol. Chem. 2001, 276, 47257-47265. (14) Guegan, C.; Vila, M.; Rosoklija, G.; Hays, A. P.; Przedborski, S. J. Neurosci. 2001, 21, 6569-6576.
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Intracellular measurements that provide new information not available from population-averaged cellular measurements are becoming increasingly important. The intracellular measurement is useful because cells in a population respond asynchronously to external stimuli.15 In addition, there is a great demand for spatially tracking many intracellular signaling pathways. Minimally invasive analysis of cellular signaling pathways that does not cause physiological or biological damage in a single cell is a requirement in intracellular measurements. To achieve the minimally invasive measurement, the physical dimensions of the probe for a single cell have to be much smaller than those of the cell. The recently developed optical nanobiosensor is a powerful tool to meet these demands.16-20 The optical nanobiosensor consists of an antibodycoated nanoprobe that can penetrate and sample individual live cells while minimally disrupting normal cellular processes such as mitosis. The intracellular components bound to the antibodies immobilized on the nanoprobe can be monitored using the evanescent field caused by a laser beam delivered to the nanoprobe. In this work, we report the detection of cytochrome c probed in a single cell using the optical nanobiosensor having mouse anticytochrome c antibodies. Following in situ binding of cytochrome c to the antibody-based nanoprobe inserted inside a single cell, an enzyme-linked immunosorbent assay,21-24 which provides the enzymatic fluorescent amplification to give high detection sensitivity, was performed with the optical nanobiosensor outside the cell for detecting cytochrome c in a MCF-7 human breast carcinoma cell treated with δ-aminolevulinic acid (5-ALA) PDT drug which induces the apoptotic pathway.25 This work demonstrates cytochrome c detection for the investigation of an apoptotic pathway at the intracellular level using the optical nanobiosensor combined with ELISA immunoassay. EXPERIMENTAL SECTION Materials. Glycidoxypropyltrimethoxysilane (GOPS) and 1,1′carbonyldiimidazole (CDI) were purchased from Aldrich (Milwaukee, WI). δ-Aminolevulinic acid and phosphate-buffered saline (PBS) were obtained from Sigma (St. Louis, MO). Acetonitrile anhydrous was received from Glen Research (Sterling, VA). Hydrochloric acid 1 N solution was purchased from Fisher Scientific Company (Fair Lawn, NJ). Mouse anti-cytochrome c (Catalog No. MS-1273-PABX) and mouse anti-cytochrome c biotin conjugate (Catalog No. MS-1273-B) were purchased from Lab Vision Corporation (Fremont, CA). Streptavidin-alkaline phosphatase conjugate was obtained from ProZyme, Inc. (San Leandro, (15) Brasuel, M.; Kopelman, R.; Miller, T. J.; Tjalkens, R.; Philbert, M. A. Anal. Chem. 2001, 73, 2221-2228. (16) Kasili, P. M.; Cullum, B. M.; Griffin, G. D.; Vo-Dinh, T. J. Nanosci. Nanotechnol. 2002, 2, 653-658. (17) Vo-Dinh, T. J. Cell. Biochem. Suppl. 2002, 39, 154-161. (18) Vo-Dinh, T.; Alarie, J. P.; Cullum, B. M.; Griffin, G. D. Nat. Biotechnol. 2000, 18, 764-767. (19) Cullum, B.; Griffin, G. D.; Miller, G. H.; Vo-Dinh, T. Anal. Biochem. 2000, 277, 25-32. (20) Cullum, B.; Vo-Dinh, T. Trends Biotechnol. 2000, 18, 388-393. (21) Bobrovnik, S. A. J. Biochem. Biophys. Methods 2003, 57, 213-236. (22) Avseenko, N. V.; Morozova, T. Y.; Ataullakhanov, F. I.; Morozov, V. N. Anal. Chem. 2002, 74, 927-933. (23) Galve, R.; Nichkova, M.; Camps, F.; Sanchez-Baeza, F.; Marco, M. P. Anal.Chem. 2002, 74, 468-478. (24) Shan, S.; Tanaka, H.; Shoyama, Y. Anal.Chem. 2001, 73, 5784-5790. (25) Hengartner, M. O. Nature 2001, 412, 27-29.
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CA). 9H-(1,3-Dichloro-9,9-dimethylacridin-2-one-7-yl)phosphate, diammonium salt (DDAO-phosphate) was purchased from Molecular Probes, Inc. (Eugene, OR). Alkaline phosphatase stabilizer and phosphatase wash solution were obtained from Kirkegaard & Perry Laboratories, Inc. (Gaithersburg, MD). The plastic-clad silica fibers were purchased from Fiberguide Industries (Stirling, NJ). Preparation of Antibody-Immobilized Optical Nanofibers. The plastic-clad silica fibers with 600-µm diameter core were pulled to make a final tip diameter of 40 nm using a laser-based micropipet pulling device (model P-2000, Sutter Instruments, Novato, CA). The pulled optical nanofibers were coated with silver metal that had a purity of 99.999% using a thermal evaporation deposition system (Cooke Vacuum Products, South Norwalk, CT). The final tip diameter was 150 nm. The nanofibers were derivatized in 10% GOPS in H2O (v/v) at 90 °C for 3 h to be silanized. The pH of the GOPS aqueous solution was adjusted to 2.8 with 1 N hydrochloric acid. The silanized nanofibers were washed in ethanol and dried overnight in a vacuum oven at 105 °C. After drying, the nanofibers were activated in a solution of saturated CDI in acetonitrile for 30 min, followed by rinsing with acetonitrile and then PBS. The nanotips were then immersed into the mouse anti-cytochrome c solution of 0.2 mg/mL (PBS solvent) and incubated overnight to allow the immobilization of capture antibodies on the nano tip. Preparation of MCF-7 Cells. Human breast cancer cell line, MCF-7 cells (Catalog No. HTB22, Rockville, MD), were grown in Dulbecco’s modified Eagle’s medium (Mediatech, Inc., Herndon, VA), which was supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY) and 1 mM L-glutamine (Gibco, Grand Island, NY). Cell cultures were grown in a humidified incubator at 37 °C in an atmosphere of 5% CO2 and 86% humidity. The cell growth was monitored daily using a microscope until the cells reached a state of confluence of 50-60%. The growth conditions were adjusted for the cells to be in log phase growth during the 5-ALA treatment and not to be so close to confluence that a confluent monolayer would form by the termination of the chemical exposure. The cells (105 cells/mL) were seeded into 60mm tissue culture dishes (Corning Costar Corp., Corning, NY), which allowed the attachment of the cells. Measurement System and Immunoassays. As shown in Figure 1, a helium-neon laser beam (Melles Griot Inc., Carlsbad, CA) at 632.8 nm was focused onto a 600-µm delivery optical fiber which was terminated with a subminiature A (SMA) connector. The antibody-immobilized tapered fiber was connected to the 600µm delivery optical fiber through the SMA connector and tied to the micromanipulator on a Nikon Diaphot 300 inverted microscope (Nikon Inc., Melville, NY) with a Diaphot 300/Diaphot 200 incubator. The manipulation system consisted of MN-2 (Narishige Co. Ltd., Tokyo, Japan) Narishige three-dimensional manipulators for coarse movement and Narishige MMW-23 three-dimensional hydraulic micromanipulators for fine movement. The Diaphot 300/ Diaphot 200 incubator maintained the cell cultures at 37 °C on the microscope stage. The mouse anti-cytochrome c immobilized nanofiber was mounted on a micropipet holder (World Precision Instruments, Inc., Sarasota, FL). The MCF-7 cells were studied as four separate groups. The first group was exposed to 0.5 mM 5-ALA at 37 °C in 5% CO2 for 3 h and then photoactivated by the
Figure 1. Schematic diagram of the experimental apparatus. A helium-neon laser beam is delivered into the delivery fiber connected to the antibody-immobilized nanoprobe. The nanoprobe is mounted on a manipulation system and inserted into a MCF-7 human breast carcinoma cell, as shown in the picture. A photomultiplier tube (PMT) was fixed in the microscope and used as a detector to measure fluorescence. To remove the laser scattering, a band-pass filter was positioned in front of the PMT.
HeNe laser beam. The laser was positioned ∼5.0 cm above the MCF-7 cells and irradiated the MCF-7 cells at a fluence of 5.0 mJ/cm2 to photoactivate 5-ALA for the apoptosis of the cells. The second group as a treated control was exposed to 0.5 mM 5-ALA for 3 h without the photoactivation. The third group as an untreated group had no exposure to either the 5-ALA or the HeNe laser beam. To demonstrate that the nanoprobe without immobilizing antibody does not have nonspecific adsorption of cytochrome c, the fourth group treated with the same conditions as the first group was probed with the nanoprobe that was not coated with the mouse anti-cytochrome c. To probe cytochrome c in a single cell, the culture dish of cells from each group was placed on the prewarmed microscope stage at 37 °C. The mouse anti-cytochrome c immobilized nanoprobe, which was mounted on the micropipet holder of a micromanipulation system, was positioned so that the nanotip was just outside of the cell to be probed using the bright-field phase contrast microscopic illumination which had total magnification of 600×. The nanoprobe was then moved into the cell membrane and extended a short way into the cytoplasm so that the nanoprobe did not penetrate the nuclear envelope. After 5 min of incubation time for binding of the intracellular cytochrome c to the antibodies on the nanoprobe, the fiber was taken out from the cell and then placed in a PBS solution. The nanoprobe was then placed in the mouse anti-cytochrome c biotin conjugate solution of 0.1 mg/mL (PBS solvent) for the cytochrome c to be incubated with the anticytochrome c biotin conjugate for 1 h. Following a rinse step with 0.5% Tween 20 in PBS, the nanoprobe was placed and incubated in a streptavidin-alkaline phosphatase conjugate solution diluted to 0.5 mg/mL in the alkaline phosphatase stabilizer for 1 h. After the nanoprobe was rinsed with the KBL phosphatase wash solution, the nanoprobe was placed in a DDAO-phosphate solution of 0.05 mM for the fluorescence measurement. Figure 2 shows a schematic diagram of ELISA immunoassay performed on the nanoprobe. A 5 mM stock solution of DDAO-phosphate was prepared by dissolving the stock solution in deionized water. When the fluorescence of the cleaved DDAO was measured, all room lights and microscope illumination lights were extinguished. The fluorescence from the nanoprobe was collected by a microscope objective and focused onto a photomultiplier tube (PMT). A bandpass filter positioned in front of the PMT was used to eliminate the laser scattering. The output signal from the PMT was read out by a universal counter and recorded in a personal computer.
Figure 2. Immunoassay on the nanoprobe. Enzyme-linked immunosorbent assay (ELISA) was peformed on the nanoprobe in order to indirectly detect the cytochrome c bound to the mouse anticytochrome c immobilized on the nanoprobe. As a result of ELISA, enzymatic product (cleaved DDAO) could be amplified.
RESULTS AND DISCUSSION Antitumor PDT using 5-ALA is based on metabolism of 5-ALA to protoporphyrin IX, which acts as a photosensitizer to provide photooxidative damage leading to apoptotic or necrotic cell death. Because the ability of the enzyme ferrochelatase to undergo metabolism of protoporphyrin IX to heme is deficient in cancer cells, the accumulation of photoporphyrin IX occurs in cancer cells.26,27 It was found that apoptosis was induced when protoporphyrin IX existed mainly in mitochondria, and necrosis occurred when protoporphyrin IX diffused to other sites, such as the plasma membrane.28 As a result of the apoptosis, cytochrome c is released into the cytoplasm, then the released cytochrome c can form a complex with dATP, apoptosis-activating factor-1 (APAF-1), and pro-caspase 9. This complex cleaves pro-caspase 9 and generates active caspase 9 that cleaves and activates pro-caspase 3.29 The resultant caspase 3 cleaves a large number of proteins that are extremely important for cell life, such as DNA fragmentation factor and poly(ADP-ribose)polymerase.30 Accordingly, the detection of (26) Battle, A. M. del C. J. Photochem. Photobiol. B 1993, 20, 5-22. (27) Peng, Q.; Warloe, T.; Berg, K.; Moan, J.; Kongshaug, M.; Giercksky, K.; Nesland, J. M. Cancer 1997, 79, 2282-2308. (28) Kriska, T.; Korytowski, W.; Girotti, A. W. Free Radical Biol. Med. 2002, 33, 1389-1402. (29) Thornberry, N. A.; Lazebnik, Y. Science 1998, 281, 1312-1316. (30) Porter, A. G.; Janicke, R. U. Cell Death Differ. 1999, 6, 99-104.
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cytochrome c is obviously a starting point for investigation of the mitochondrial apoptotic pathway at the intracellular level. In this work, the detection of cytochrome c using the nanoprobe was performed with ELISA immunoassay. This technique can be very useful when probing intracellular components whose amounts are not large in a cell and whose detection sensitivities are relatively low. This is because the enzymatic reaction between DDAO-phosphate and streptavidin-alkaline phosphatase conjugate in the immunocomplex provides a large amount of the cleaved DDAO fluorescence. DDAO-phosphate, which is nonfluorescent, enables a low background. This enzymatic amplification was proven to provide a much more sensitive detection of the target antigen, as compared to the immunoassay without enzymatic amplification. The optical nanobiosensor detection is based on excitations of cleaved DDAOs using an evanescent field. Due to the 40-nm diameter of the nanoprobe, the delivered laser beam through the delivery optical fiber cannot penetrate the nanotip. In this diffraction-limited condition, the evanescent field, which allows the laser beam energy to be transmitted in the form of an interfacial leaky surface mode, becomes the only excitation source for the cleaved DDAO. In order for the cleaved DDAO to be excited by the evanescent field, the cleaved DDAO has to diffuse into the probing area where excitations are caused by the evanescent field. This is because only molecules in close proximity to the boundary interface of the nanoprobe can be excited. The laser beam intensity decays exponentially from the boundary interface of the nanoprobe. The cleaved DDAO then repeats excitations and emissions in the probing area by the evanescent field during the sampling time. Optical detection based on evanescent field excitation has the advantage of effectively reducing the contribution of laser scattering to the background signal. In addition, the only choice that allows a minimally invasive intracellular measurement without disrupting normal cellular activities seems to be the nanobiosensor; however, due to its nanoscale size, the nanotip does not provide a large probing area. This means only a small amount of the fluorescent antigen immobilized on the nanotip can be excited. As a result, in the nanobiosensor, detection sensitivity can be a tradeoff with the advantage of minimally invasive intracellular measurement. Accordingly, the indirect detection of protein using an ELISA immunoassay to provide a lot of the enzymatic products is an efficient way to improve the detection sensitivity for the nanobiosensor. Figure 3 shows results of cytochrome c detection obtained from cells of four different groups using nanoprobes. The relative intensity of each group was obtained from four replicate nanoprobes, and the average intensity was plotted. Error bars represent the (standard deviation (SD) of four measurements where each measurement has 40 different intensity values obtained using a nanoprobe. Signal A represents the background signal obtained from cells untreated with 5-ALA. Signal B represents the relative fluorescence intensity obtained from cells treated with 5-ALA but without photoactivation. Signal C represents the relative fluorescence intensity acquired from cells treated with 5-ALA PDT. Signal D represents the background signal obtained from cells treated with 5-ALA PDT and probed by the nanoprobe without immobilizing antibody. Compared to the relative intensities obtained from cells which are treated with 5-ALA without the photoactivation and untreated with 5-ALA, 5-ALA PDT-treated cells show a much 2594 Analytical Chemistry, Vol. 76, No. 9, May 1, 2004
Figure 3. Results of fluorescence measurements using the nanoprobes in different cells with or without photodynamic therapy (PDT) using D-aminolevulinic acid (5-ALA): (A) background (not treated with 5-ALA), (B) treated with 5-ALA without photoactivation, (C, D) treated with 5-ALA PDT. Antibody-coated nanoprobes were used for A, B, and C. In the case of D, the nanoprobe without immobilizing antibody was used to probe the cells.
larger fluorescence intensity of cytochrome c. The cells treated with 5-ALA without the photoactivation show the relative intensity, which is not enough to identify cytochrome c. Because the probing area under the influence of the evanescent field is extremely small, the interference level should be extremely low. Accordingly, the above results clearly represent the detection of cytochrome c released into the cytoplasm from the mitochondria after 5-ALA PDT by the nanoprobe. Signal D demonstrates that the nanoprobe does not have nonspecific adsorption of the cytochrome c when the nanoprobe is inserted into cells treated with 5-ALA PDT. The measurement showed excellent reproducibility of nanoprobe-tonanoprobe measurements. The calculated standard deviation was ∼8%. Highly reproducible fabrication of the nanoprobe is thought to contribute mainly to the high degree of reproducibility of the nanoprobe-to-nanoprobe measurements. CONCLUSION Cytochrome c detection in a single cell using the nanoprobe was demonstrated in this work. Due to the importance of the role of cytochrome c in apoptosis, our results open a way to investigate the apoptotic pathway at the single cell level. The nanoprobe could allow the minimally invasive probing of cytochrome c in a MCF-7 cell treated with 5-ALA PDT activation and provide high fluorescent intensity compared to the background signal. The nanoprobe combined with ELISA immunoassay allowed high sensitivity detection due to the enzymatic amplification of target fluorescence on the nanoprobe. This technique should be very useful for detection of other intracellular components whose amounts are not large in a cell. ACKNOWLEDGMENT This research is sponsored by the Office of Biological and Environmental Research, U.S. Department of Energy under contract DE-AC05-960R22464 program with UT-Battelle. In addition, J. M. Song and P. M. Kasili are supported by an appointment to the Oak Ridge National Laboratory Postdoctoral Research Associates Program administered by the Oak Ridge National Laboratory and Oak Ridge Institute for Science and Education. Received for review October 31, 2003. Accepted February 27, 2004. AC0352878
