Multiplex Targeting, Tracking, and Imaging of Apoptosis by

Bioconjugate Chem. 2007, 18, 1155−1162

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Multiplex Targeting, Tracking, and Imaging of Apoptosis by Fluorescent Surface Enhanced Raman Spectroscopic Dots Kyeong Nam Yu,†,# Sang-Myung Lee,‡,# Ji Yun Han,§,# Hyunmi Park,§ Min-Ah Woo,† Mi Suk Noh,† Soon-Kyung Hwang,† Jung-Taek Kwon,† Hua Jin,† Yong-Kweon Kim,| Paul J. Hergenrother,⊥ Dae Hong Jeong,*,§ Yoon-Sik Lee,*,‡ and Myung-Haing Cho*,† Laboratory of Toxicology, College of Veterinary Medicine, School of Chemical and Biological Engineering, Department of Chemistry Education, and School of Electrical Engineering and Computer Science, Seoul National University, Seoul 151-742, Korea, and Department of Chemistry, University of Illinois, Urbana, Illinois 61801. Received January 11, 2007; Revised Manuscript Received April 26, 2007

We have developed multifunctional fluorescent surface enhanced Raman spectroscopic tagging material (F-SERS dots) composed of silver nanoparticle-embedded silica spheres with fluorescent organic dye and specific Raman labels for multiplex targeting, tracking, and imaging of cellular/molecular events in the living organism. In this study, F-SERS dots fabricated with specific target antibodies (BAX and BAD) were employed for the detection of apoptosis. The F-SERS dots did not show any particular toxicity in several cell lines. The F-SERS dots could monitor the apoptosis effectively and simultaneously through fluorescent images as well as Raman signals in both cells and tissues with high selectivity. Our results clearly demonstrate that F-SERS dots can be easily applicable to multiplex analysis of diverse cellular/molecular events important for maintaining cellular homeostasis.

INTRODUCTION The current development of nanotechnology has led to a strong motivation for the connection between nanoscience and biology. In fact, scientific interests between nanotechnology and biology are based on the perceptions that nanotechnology offers biology new tools and that biology offers nanotechnology access to new types of functional nanosystems (1). In this regard, recent efforts in such nanobio fusion technology have been partly focused on the development of nonradioactive tagging technology for the investigation of complex interplay of biomolecules, because it may provide safe, specific, and stable alternatives. Up to now, several types of tagging materials have been developed for the application into biology. For example, fluorescence-based materials and semiconductor quantum dots have been used widely. However, some intrinsic problems such as broad emission profiles leading to peak overlapping in fluorescence/emission tagging, difficulties in surface modification, and potential safety issues in quantum dots have impeded the practical application into biology (2). Apoptosis, a form of programmed cell death, plays a critical role in the maintenance of cells by providing a controlled cell deletion to a balanced cell proliferation. Apoptosis is characterized by specific morphological changes such as nuclear frag* Corresponding authors. Dae Hong Jeong, Ph.D., Department of Chemistry Education, Seoul National University, Seoul 151-742, Korea, E-mail: [email protected]. Yoon-Sik Lee, Ph.D., School of Chemical and Biological Engineering, Seoul National University, Seoul 151-742, Korea, E-mail: [email protected]. Myung-Haing Cho, D.V.M., Ph.D., Laboratory of Toxicology, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Korea, E-mail: [email protected]. † College of Veterinary Medicine, Seoul National University. ‡ School of Chemical and Biological Engineering, Seoul National University. § Department of Chemistry Education, Seoul National University. | School of Electrical Engineering and Computer Science, Seoul National University. ⊥ University of Illinois. # These authors contributed equally to this work.

mentation, membrane blebbing, and cell shrinkage. At the molecular level, apoptosis represents a collection of intricate pathways with many proteins actively participating in activities from cell signals. Failure of tumor cells to undergo apoptosis translates into malignant potential (3). Therefore, multiplex detection of several key targets important for apoptosis should provide critical clues for cells/tissues to cope with diverse abnormal cellular stimulation. Up to the present, diverse bioapplications have emerged from several nanoparticles. The addition of fluorescence properties to nanoparticles may offer new potential for in Vitro and in ViVo imaging (4). There have been several reports on tagging strategy using Raman spectroscopy featuring the narrow band structure for multiplex biological targeting (5-10). However, such previous reports have still not reached a stage for multiplex detection at once. Recently, our multidisciplinary research group reported a simple and highly reproducible procedure to generate a new type of Raman tag called surface-enhanced Raman spectroscopic dots (SERS dots) in which Raman scatterings of the molecules adsorbed thereon are dramatically enhanced (2). Current study, therefore, has been motivated by the exploratory use of unique advantages of fluorescence-based tracking and imaging of the site of interest as a first step and then subsequent characterization by SERS-based multiplex reading of signals important for cellular/molecular changes. For this purpose, we have developed specific fluorescent SERS dots (F-SERS dots) composed of silver nanoparticle-embedded silica spheres with fluorescent dye and specific Raman labels. Here, we report that F-SERS dots can be employed for multiplex targeting, tracking, and imaging of apoptosis at once in cells/tissues. This concept of multifunctional F-SERS dots may serve as a platform technology for the diagnostic as well as therapeutic application in the future.

EXPERIMENTAL PROCEDURES Preparation of F-SERS Dot. Silica nanoparticles of ca. 120 nm size were prepared by the Sto¨ber method (11). A 1 mL portion of ammonium hydroxide (27%) was added into 78 mL

10.1021/bc070011i CCC: $37.00 © 2007 American Chemical Society Published on Web 06/29/2007

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ethanol (95%) and 10 mL H2O, and then 5 mL (22.4 mmol) of tetraethylorthosilicate (TEOS) was added into the solution. The solution was stirred with a magnetic bar vigorously for 12 h at 25 °C. The resulting silica colloids were centrifuged and washed with ethanol several times. To obtain mercapto-functionalized silica nanoparticles (MSNP), 300 mg portion of silica nanoparticles was dispersed in ethanol, and then 150 µL of 3-mercaptopropyltrimethoxysilane (MPTS) and 300 µL of ammonium hydroxide were added to the colloidal solution. The mixture was stirred with a magnetic bar vigorously for 12 h at 25 °C again. The resulting MSNPs were centrifuged and washed with ethanol several times to remove excess reagents. Silver nanoparticles were embedded on the surfaces of silica nanoparticles by the Sn2+-reduction method (12). At first, 160 mg of silica nanoparticles were dispersed in water/ethanol solution (15 mL/15 mL), and then 100 µL of trifluoroacetic acid (TFA) and 100 mg of SnCl2 were added. The mixture was stirred vigorously for 1 h at 25 °C. The resulting silica nanoparticles were centrifuged and washed with ethanol (20 mL × 3). The Sn2+-coated silica nanoparticles were redispersed in 250 mL of water, and 50 mL of 6 mM AgNO3 was added dropwise under vigorous magnetic stirring at 25 °C. The dispersion was centrifuged and washed with water (30 mL × 2 times) and ethanol (30 mL × 2 times) to remove excess reagents. In order to introduce the dual signals of Raman spectroscopy and fluorescence to the silica nanoparticles, 4-aminothiophenol (ATP) and 4-mercaptotoluene (MT) were used as Raman spectroscopic coding chemicals, and fluoresceinisothiocyanate (FITC) and Alexafluoro 647 (AF647) as fluorescent tagging chemicals. First of all, Raman chemicals were introduced together with MPTS allowing a self-assembled monolayer on the silver surface of silica nanoparticles. Raman chemicals (10 mL of 5 mM in ethanol) and MPTS (10 mL of 50 mM in ethanol) were mixed and added into a 50 mg portion of silverembedded silica nanoparticles. The dispersion was stirred vigorously for 1 h at 25 °C. The colloids were centrifuged and washed with ethanol (20 mL × 3 times) and water (20 mL). These silica nanoparticles were dispersed in 20 mL of water, and 30 µL of aqueous sodium silicate (27%) was added into the dispersion and stirred vigorously for 12 h at 25 °C (13). The resulting colloids were centrifuged and washed with water (20 mL × 3) and ethanol (20 mL) to remove excess reagents. In the next step, a fluorescent layer was introduced onto silica nanoparticles labeled with Raman signals. For this process, the conjugates of 3-aminopropyltriethoxysilane (APS) and fluorescence dyes were synthesized. In the case of FITC, FITC (10 µL of 8 mM in DMSO) was added into the APS (100 µL of 19.2 mM in ethanol), and the resulting solution was stirred for 6 h at 25 °C. Then, the FITC-APS conjugate, 20 µL of TEOS, and 40 µL of aqueous ammonium hydroxide (25%) were added into the 20 mg portion of silica nanoparticles with Raman labels. The mixture was stirred vigorously for 12 h at 25 °C. The resulting nanoparticles (F-SERS dots) were centrifuged and washed with ethanol thoroughly. Thereafter, the surface of F-SERS dots was modified with a hydrophilic spacer, Nfluorenylmethylcarbonyl-NH-triethylene glycol-NH-COCH2CH2COOH (Fmoc-TEG-COOH). This spacer was synthesized from 4,7,10-trioxa-1,13-tridecanediamine by the literature procedure (14). F-SERS dots (20 mg) were dispersed in N,Ndimethylformamide (DMF) and aliquots (0.1 mmol) of FmocTEG-COOH, N,N-diisopropylcarbodiimide (DIC), N-hydroxybenzotriazole (HOBT), and diisopropylethylamine (DIEA) were added into the dispersion, consecutively. The mixture was shaken vigorously for 6 h at 30 °C. After the coupling reaction, Fmoc protecting groups were removed from the spacer on F-SERS dots by 20 vol % piperidine/DMF solution for 30 min

Yu et al.

at 25 °C. The resulting triethylene glycol (TEG) grafted F-SERS dots (F-SERS dots/TEG-NH2) were centrifuged and washed with ethanol thoroughly. Antibody Conjugation onto F-SERS Dots. F-SERS dots/ TEG-NH2 were treated with 0.1 mmol of 3-maleimidopropionic acid N-hydroxysuccinimide ester in DMF for 2 h at 25 °C. The resulting nanoparticles were centrifuged and washed with DMF, water, and PBS (100 mM, pH 7.4). Before antibody conjugation, full-bodied antibody was reduced and fragmented into two parts (15). This procedure was applied to bcl2-associated death promoter (BAD) and bcl2-associated X protein (BAX) antibodies. Antibody (20 µL, 100 µg/mL) was pretreated with 5 µL of ethylenediaminetetraaceticacid (EDTA, 0.5 M, pH 8.0), and then followed by adding 2 µL of 2-mercaptoethylamine (MEA, 0.312 mmol) and 5 µL of 0.5 M EDTA in 200 µL of PBS (pH 7.4). The mixture was incubated at 37 °C for 90 min. The resulting half-fragmented antibody was purified by gel filtration using Sephadex G-25 and then used for conjugation without additional handling. The 200 µL of reduced antibody solution was added into 10 mg portion of maleimide-modified F-SERS dots dispersed in PBS (100 mM, pH 7.4). The dispersion was incubated at 25 °C for 2 h. The F-SERS dots/TEG-antibody conjugates were centrifuged and washed with PBS containing 0.1 wt % Tween 20 (1 mL × 3 times), and PBS (1 mL × 2 times), consecutively. Annexin V Conjugation onto the F-SERS Dots. The amine termini of F-SERS dots/TEG-NH2 were modified to carboxyl termini for immobilizing Annexin V. F-SERS dots/TEG-NH2 (10 mg) was dispersed in 10 mM succinic anhydride and 10 mM DIEA in DMF, and shaken for 2 h at 30 °C. They were centrifuged and washed with DMF (1 mL × 3 times), H2O (1 mL × 3 times). They were redispersed in 2-(N-morpholino)ethanesulfonic acid buffer (MES, 25 mM, pH 6.0), followed by adding 50 µL of 5 mg/mL 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDC) and 50 µL of 5 mg/ mL N-hydroxysuccinimide (NHS) in MES buffer. The dispersion was incubated for 1 h at 25 °C. The activated nanoparticles were washed with MES (1 mL × 3 times) and redispered in MES buffer. Immediately, 20 µL of 100 µg/mL Annexin V was added into the activated nanoparticles mixture and incubated for 2 h at 25 °C. The resulting F-SERS dots-Annexin V conjugates were treated with 10 mM ethanolamine in PBS for 30 min for quenching the unreacted activating sites, and washed with PBS containing 0.1 wt % Tween 20 (1 mL × 3 times), and PBS (1 mL × 2 times), consecutively. Cell Culture and Viability Assay. Normal lung epithelial cells (WI-38), and breast mammary gland epithelial cancer cell (SK-BR3) were obtained from Korea Cell Line Bank (Seoul, Korea), and lung squamous carcinoma cells (H226) were obtained from ATCC (American Type Culture Collection). H226 and SK-BR3 cells were cultured in RPMI-1640 with 10% fetal bovine serum (FBS, Hyclone, Logan, UT), and WI-38 cells were cultured in MEM with 10% FBS. Cultures were maintained at 37 °C in 5% CO2 atmosphere. Effects of F-SERS dots on viability of several cell lines were determined using a CCK-8 assay kit according to manufacturer’s protocol (Dojindo, Kumamoto, Japan). Briefly, cells were seeded at 5 × 104 cells on each well and treated with F-SERS dots for 24 h. Samples were analyzed in triplicate. Results were expressed as a percentage of viable cells of untreated control. Detection of Apoptosis in Cells. Cells were treated with 0.1 µM staurosporine (STS, Sigma-Aldrich, St. Louis, MO) for 24 h in order to induce apoptosis. Our preliminary works indicate that STS at this concentration is sufficient to induce significant apoptosis in diverse cell lines used in this study. Annexin V is a calcium-dependent phospholipid-binding protein with high affinity for phosphatidylserine (16). Therefore, Annexin V-

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Detection of Apoptosis by Fluorescent SERS Dots

Figure 1. Synthetic scheme of F-SERS dots. Preparation of fluorescent Raman active silica nanoparticle and surface modification of the F-SERS dots with antibody or Annexin V (A). HR-TEM images of silica nanoparticles embedded with silver nanoparticles by Sn2+-reduction method (B-a), after silica shell encapsulation, and then amino-functionalized F-SERS dots labeled with ATP and FITC (B-b) and high-magnification image (B-c).

conjugated F-SERS dots were employed to detect the translocation of phosphatidylserine residues from the inner layer to the outer layer of the plasma membrane. Briefly, cells were grown on chamber slides (Nalge Nunc, Naperville, IL) and treated with STS for 24 h. Then, Annexin V-conjugated F-SERS dots were added and then incubated for 6 h. Slides were then washed with PBS and fixed in 4% paraformaldehyde for 10 min. The slides were examined using a confocal laser scanning microscope (CLSM, Nikon, Tokyo, Japan). Detection of Apoptosis in Murine Lung Cancer Tissues. Apoptosis study on tissue with the F-SERS dots was carried out onto PAC-1 treated murine lung cancer tissues. Our previous works demonstrated that PAC-1 induced apoptosis significantly through activation of procaspase-3 to caspase-3 in the lungs from mice carrying lung cancer (17). Therefore, we adopted the lung samples for current study. The immunohistochemistry on lung tissue was performed with BAX and BAD antibodies with a slightly modified method from Jin et al. (18). For the immunoassay with antibody-conjugated F-SERS dots, sample slides were processed according to the same procedures of immunohistochemistry until the blocking process. At the blocking step, F-SERS dots diluted in PBS were added into tissue slides. The slides were incubated for 24 h under dark condition. After incubation, the site of apoptosis was initially identified by fluorescence signals, and then, multiplex targeting was characterized by Raman analysis using a confocal Raman system (LabRam 300, JY-Horiba, Edison, NJ) equipped with an optical microscope (Olympus, Tokyo, Japan). In this system, the Raman scattering signals were collected in a 180° scattering geometry and detected by a spectrometer equipped

with a thermoelectrically cooled CCD detector. A 514.5 nm laser line from Ar ion laser (35-MAP-321, Melles Griot, Carlsbad, CA) was used as an excitation source. The strong Rayleigh line was eliminated by a holographic notch filter placed in the collection path. Raman scattered light was collected with a 100× objective microscope (0.95 NA, Olympus). Approximately less than 1 mW of laser irradiation was used to excite samples for Raman measurements. The signal collection time was 30 s.

RESULTS Synthesis of Annexin V-Conjugated F-SERS Dots. The silica nanoparticles (ca.120 nm) as a supporting material were successfully synthesized (Figure 1A). As shown, silver nanoparticles were produced by the Sn2+-reduction method and embedded onto the silica nanoparticles, which were fabricated with Raman chemicals such as ATP or MT and MPTS. The Ag nanoparticle-embedded silica nanoparticles, which contained Raman chemicals, were overcoated with silica shell. The nanoparticles were then treated with APS-organic dye (FITC or AF647)/TEOS under basic conditions to form F-SERS dots. In order to conjugate F-SERS dots with half-fragmented antibody (BAD and BAX), the surface of F-SERS dots was modified with hydrophilic spacer (Fmoc-TEG-COOH). The halfantibodies were prepared by reducing disulfide bonds of the antibody heavy chain using MEA. Also, in the case of Annexin V conjugation for specific targeting to phosphatidylserine, the F-SERS dots were further treated with APS for amine termination and then coupled with the carboxylic group of Annexin V


Figure 2. Cell viability assay for potential cytotoxicity of F-SERS dots in various cell lines (WI-38, SK-BR3, and H226). Each cell seeded at 5 × 104 cells/cm2 was treated with F-SERS dots at different concentrations and incubated for 24 h. All cell viability was expressed as a percentage of viable cells with untreated control cells set at 100%.

leading to amide bond formation. Figure 1B exhibited that the silver nanoparticles were successfully embedded and overcoated with silica shell. The thickness of overcoated silica shell was about 3 nm by high-resolution transmission electron microscope (HR-TEM). Cytotoxicity Study of F-SERS Dots. The cytotoxicity of the F-SERS dots in various cell lines such as WI-38, SK-BR3, and H226 cells was evaluated (Figure 2). All cell lines were incubated for 24 h at 37 °C, 5% CO2, and the FSERS dots containing media were discarded for CCK-8 assay. Our data strongly suggest that F-SERS dots fabricated in this study are not toxic, because cell viabilities remained over 90%. Specific Targeting with Annexin V-Conjugated F-SERS Dots in the Apoptotic Cells. To demonstrate the specific targeting of phosphatidylserine with Annexin V-conjugated F-SERS dots which were tagged with ATP as Raman chemical and anchored with AF647 (red color), we performed an assay with the cells pretreated with STS. At first, in order to confirm the specific targeting ability, FITC (green color) labeled Annexin V was monitored within STS-treated SK-BR3 cells by CLSM

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Figure 3. CLSM analysis of specific phosphatidylserine targeting with Annexin-V conjugated F-SERS dots in SK-BR3 cells. (A-a,b) Normal untreated cell. (A-c,d) STS treated cell. (B-a,b) Highmagnification images (600×) from apoptotic cell. (B, bottom) z-Section images of apoptosis cells (scanned interval of 0.3 µm). A-a, A-c, and B-a are bright field images; A-b, A-d, and B-b are fluorescence images.

and FACS (see the Supporting Information, SI-Figure 1). We confirmed that FITC-labeled Annexin V could target the apoptosis successfully, because green color was only found in the outer cell membrane (SI-Figure 1A-c), while such staining was not observed in control cells (SI-Figure 1A-a). Such apoptosis was also confirmed by flow cytometry analysis (SIFigure 1B), thus suggesting that used STS was sufficient to induce apoptosis. Figure 3A showed that the Annexin Vconjugated F-SERS dots could recognize the apoptosis in outer plasma membrane where Annexin V bound to phosphatidylserine selectively. In comparison to STS-treated cells (Figure 3A-d), such red signals were not observed in control cells (Figure 3A-b). Please note that the red fluorescence from F-SERS dots was detected at most of the apoptotic cells. In Figure 3B, high-magnification z-section images of cells exhibited clear evidence of reddish nanoparticle location at the outer cell membrane. In contrast, F-SERS dots without Annexin V were internalized into the cells, and such uptake of nonmodified F-SERS dots into the cells was clearly observed in the study



Figure 4. SERS analysis of specific phosphatidylserine targeting with Annexin V conjugated F-SERS dots. A and B represent a control normal cell and a STS-treated cell, respectively. A-a and B-a are bright field optical images of normal and treated cells, respectively. A-b and B-b are SERS intensity maps for 1431 cm-1 Raman band, and A-c and B-c depict SERS spectra at several positions indicated as white dots in parts A-b and B-b, respectively. The whole area was scanned by 10 × 10 steps, and Raman signals were collected for 30 s each step.

by CLSM and Raman analysis (SI-Figure 2). According to z-section scanning of CLSM, we knew that the internalized F-SERS dots were primarily located at the cytoplasm of the cells. The F-SERS dots were not present in the nucleus, which was confirmed by nucleus staining with DAPI (SI-Figure 2A). Raman spectra were detected in all areas of fluorescence (SIFigure 2B). Together, F-SERS dots with Annexin V could detect the apoptosis effectively through specific binding to phosphatidylserine in outer plasma membrane. Since the fluorescence and SERS spectra of F-SERS dots were spectrally well-separated, we could obtain the SERS spectra from cells emitting fluorescence lights (Figure 4). When the cells were not treated with STS, the SERS signals from the F-SERS dots were not detected in most of cell region (Figure 4A). On the other hand, the SERS signals were clearly observed in the cells treated with STS (Figure 4B). Figure 4A-b,B-b demonstrated the SERS intensity maps for the 1431 cm-1 Raman band of ATP corresponding to the regions shown in Figure 4A-a,B-a images, respectively. The same scales of the brightness for both SERS intensity maps were used to visualize their different intensities. White blocks represent stronger SERS signals compared to black blocks. Selected SERS spectra for

cells with and without STS treatment are shown in Figure 4A-c and B-c, respectively. Most regions of the cell without STS exhibited no SERS signals, but some parts depicted SERS signals with very weak intensity, such as at A9 and I2 positions in Figure 4A-b reflecting a noncorrelated distribution with cell morphology presumably due to nonspecifically bound F-SERS dots. However, the cell treated with STS exhibited a highly correlated distribution of strong intensity with cell morphology as well as strong SERS spectra as shown in Figure 4B-c. Detection of Multiple Targets of Apoptosis in the Murine Lung Tissues with F-SERS Dots at once. In order to locate the expressed sites of BAX and BAD proteins on the tumor tissue, an immunoassay with F-SERS dots was performed in the murine lung cancer tissues adopted from previous study (17). Please note that brown color indicates the apoptotic proteins such as BAX (Figure 5A upper left (×200), lower left (×400)) and BAD (Figure 5A upper right (×200), lower right (×400)). The white arrows are position markers in sequential slices. On the basis of the effective searching the sites of apoptosis by fluorescence signals (representative white square in Figure 5), we tried to characterize the apoptosis through Raman scattering. The green blocks in Figure 6a indicate the SERS intensity of


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Figure 5. Immunohistochemistry and CLSM image of BAX/BAD protein on PAC-1 induced apoptosis in murine lung tissue. (A) Immunohistochemistry images of BAX (left)/BAD (right) and low magnification (up, 200×)/high magnification (down, 400×). (B) Fluorescent images of F-SERS dots (400×). White square indicates the same areas scanned by Raman analysis. White arrows are the position markers.

the 384 cm-1 Raman band of MT (signal of BAX), and the red blocks in Figure 6b indicate the SERS intensity of the 1431 cm-1 Raman band of ATP (signal of BAD). Figure 6c is a merged image of both images to illustrate the distributions of BAX and BAD proteins in the tissue. In most parts, occurrences of BAX and BAD were similarly distributed with some exceptions such as positions C and D marked in Figure 6c. The representative SERS spectra from the tissue are depicted in Figure 6d. As illustrated, F-SERS dots were able to detect the multiple targets of apoptosis in the murine lung cancer tissue at once.

DISCUSSION Nanotechnology has been applied to various biological areas such as specific targeting, cell sorting, and fluorescent or magnetic imaging. The increased application of nanotechnology can, however, be either positive or negative or a mix of both (19). Therefore, the biocompatibility of nanomaterials is a very important factor. In this regard, we performed a cytotoxicity study of F-SERS dots on several cell lines (Figure 2). On the basis of our results, F-SERS dots seemed to be nontoxic, thus suggesting that F-SERS dots may be used as a biocompatible tool. Analysis of current literature suggests that combining several biomarkers improves sensitivity and specificity of many diseases dramatically (20). Therefore, a multimarker approach

for detection and monitoring of disease status should have a great potential in the future (21). In this regard, several research groups have successfully linked fluorescent nanoparticles to peptides, proteins, and oligonucleotides (22). Quantum dots are fluorescent nanoparticles and emit narrow symmetrical emission peaks with minimum overlap between spectra, thus allowing unique resolution of their spectra. These key advantages make it possible to label molecular targets by quantum dots both in Vitro and in ViVo (23). However, the use of quantum dots may be limited by the difficulty in surface modification and the potential toxic effects of the heavy metal core (24). The use of organic fluorescent molecules as tags for the probe of interest also has some limitations such as influence on fluorescence intensity from environmental molecules, broad emission profiles, and photobleaching problems (25). SERS has gaining attention because it is another sensitive method for spectroscopic detection of multiple targets in aqueous and solid samples (26). The benefit of SERS and nanoparticles in terms of sensitivity, specificity, and selectivity has been previously proven by our previous works (2). In this study, F-SERS dots were successfully synthesized by the wellestablished procedure, and the size and the shape were wellcharacterized by TEM (Figure 1). Moreover, the modified F-SERS dots could provide a rapid and specific way to monitor the apoptosis in both cells and tissues by simultaneous use of



Figure 6. SERS intensity maps indicating distribution of BAX and BAD proteins on the tumor tissue. The maps were produced using the baselinecorrected intensities of the 384 cm-1 Raman band of MT corresponding to BAX proteins (a) and the 1431 cm-1 Raman band of ATP corresponding to BAD proteins (b), respectively. Part c represents the merged figure of parts a and b to illustrate the distribution of both apoptotic proteins.

fluorescence and Raman signals (Figures 3-6). Interestingly, Annexin V-conjugated F-SERS dots remained in the outer plasma membrane due to the selective binding to phosphatidylserine (27), while F-SERS dots without Annexin V were translocated into the cells; thus, we could confirm the specificity and selectivity of F-SERS dots (Figure 3, and Supporting Information). Furthermore, multiplex targeting under the guide of tracking and imaging of F-SERS dots were clearly demonstrated in the study done with murine lung cancer tissue samples. In a classical study of immunohistochemistry, it is difficult to detect multiple targets in one time process. Moreover, immunohistochemistry takes a relatively long time (more than 72 h). In contrast to such time-consuming immunohistochemistry, however, multiplex targeting of BAX/BAD protein expression by F-SERS dots with different encoding chemicals was simple, accurate, effective, and reproducible (Figures 5 and 6). Immunoassay with F-SERS dots just requires one sample due to the multifunction of fluorescence and Raman signals. Taken together, the simplicity and specificity of FSERS dots for the simultaneous multidetection of apoptosis at cells or tissues were proven clearly. In conclusion, our results strongly demonstrate that F-SERS dots may be applicable to multiplex analysis of diverse cellular/molecular events important for maintaining cell/tissue homeostasis in the future.

ACKNOWLEDGMENT This work is partly supported by NCI-NCRC program of KOSEF. Supporting Information Available: Additional imaging as described in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

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