Aptamer-Guided Silver–Gold Bimetallic Nanostructures with Highly

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Aptamer-Guided Silver−Gold Bimetallic Nanostructures with Highly Active Surface-Enhanced Raman Scattering for Specific Detection and Near-Infrared Photothermal Therapy of Human Breast Cancer Cells Ping Wu, Yang Gao, Hui Zhang, and Chenxin Cai* Jiangsu Key Laboratory of New Power Batteries, College of Chemistry and Materials Science, Nanjing Normal University, Nanjing 210097, People’s Republic of China S Supporting Information *

ABSTRACT: The aptamer (S2.2)-guided Ag-Au nanostructures (aptamer−Ag-Au) have been synthesized by photoreduction and validated by ultraviolet−visible light (UV−vis) spectra and transmission electron microscopy (TEM) images. Differential interference contrast (DIC), fluorescence, and TEM images, and surface-enhanced Raman scattering (SERS) spectra indicated that the aptamer−Ag-Au nanostructures can target the surface of human breast cancer cells (MCF-7) with high affinity and specificity. This targeting is completed via the specific interaction between S2.2 aptamer (a 25-base oligonucleotide) and MUC1 mucin (a large transmembrane glycoprotein, whose expression increased at least 10-fold at MCF-7 cells in primary and metastatic breast cancers). However, the nanostructures cannot target HepG2 (human liver cancer cells) or MCF-10A cells (human normal breast epithelial cells), because these cells are MUC1-negative expressed. Moreover, the synthesized nanostructures exhibited a high SERS activity. Based on these results, a new assay for specifically detecting MCF-7 cells has been proposed. This assay can also discriminate MCF-7 cells from MCF-10A cells and different cancer cell lines, such as HepG2 cells. In addition, the aptamer−AgAu nanostructures have a high capability of adsorpting near-infrared (NIR) irradiation and are able to perform photothermal therapy of MCF-7 cells at a very low irradiation power density (0.25 W/cm2) without destroying the healthy cells and the surrounding normal tissue. Therefore, the proposed assay is significant for the diagnosis of tumors in their nascent stage. The synthesized nanostructures could offer a protocol to specifically recognize and sensitively detect the cancer cells, and would have great potential for application in the photothermal therapy of the cancers.

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cancer diagnosis through a biopsy of breast tissue in advanced stages of the disease. These methods are not sensitive enough to detect the breast cancer in its nascent stage. Testing for cancerous cells is a very effective method to diagnosis of tumors, especially in their nascent stage, since the occurrence and development of cancer are closely related to the change of cells, such as cell surface components, cell proliferation, and cell differentiation.3,4 However, the level of cancerous cells within the biological system at the early stages of cancer is particularly too low to be detected by the present available methods. Therefore, the development of novel approaches to detect cancer cells as sensitively and selectively as possible is in crucial need. We report an assay for detecting human breast cancer cell (MCF-7) at the single-cell level, based on the specific interaction between the MUC1 mucin on the surface of MCF-7 cells and its aptamer.

reast cancer is, by far, the most frequent cause of cancer death in women.1,2 In 2008, an estimated 458 000 women died from the disease, about 1 380 000 new cases of breast cancer were diagnosed, ranking it the second-most-frequently diagnosed cancer and accounting for ∼10.9% of all malignancies.2 The majority of these cases are treatable and curable, much of that treatability and curability significantly depends on early detection, early diagnosis, and early treatment. Five-year survival for localized and regional breast cancer is 98% and 84%, respectively, if it is detected early. However, when the cancer has spread to distant organs, this value will drop to 23%.1,2 Therefore, early detection of breast cancer is of paramount importance for diagnosis, treatment, and improvement of survivorship. Current detection methods for breast cancer include mammogram, breast ultrasound, breast MRI (magnetic resonance imaging), and breast biopsy, etc. Mammography is the first step for breast cancer screening and diagnosis; however, it is less accurate in patients with dense breast tissue, implants or other factors that result in complex breast tissue. Breast biopsy is the final confirmation of a breast © 2012 American Chemical Society

Received: March 25, 2012 Accepted: August 27, 2012 Published: August 27, 2012 7692

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(UV) light irradiation, using a standard portable 6-W UV lamp. The color of the solution changed from colorless to pink gradually, indicative of the formation of Ag nanostructures on the aptamer (aptamer-Ag).19 An exposure time of 30 min was used because longer exposure times led to the agglomeration of the produced Ag nanoparticles. Afterward, 1.2 mL of NaAuCl4 solution (1 mM) was added to the aptamer-Ag nanostructures solution with a AuCl4−:Ag+ molar ratio of 4. Photoreduction then was performed again by exposure to UV light for 30 min. The color of the mixture changed to blue under the irradiation, indicating the formation of Ag-Au nanostructures on aptamer (aptamer−Ag-Au).20 UV−vis spectrum was recorded on a Cary 5000 UV−vis spectrophotometer (Varian, USA) at each step, to monitor the formation of nanostructures. Transmission electron microscopy (TEM) images of the synthesized nanostructures were recorded on a JEOL Model 2010 TEM microscope operated at an accelerating voltage of 120 kV (JEOL, Japan). High-angle annular dark-field scanning TEM (HAADF-STEM) images and element analysis mapping were carried out on a Technai G2 F30 S-Twin TEM microscope operated at 200 kV (FEI, USA). Cell Culture. MCF-7 cells and HepG2 cells (human liver cancer cells) were obtained from the Cell Center of the Chinese Academy of Sciences (Shanghai, China). MCF-10A cells (human normal breast epithelial cells) were obtained from ATCC (Rockville, MD). MCF-7 and HepG2 cells were cultured at 37 °C in RPMI 1640 medium supplemented with 10% premium fetal bovine serum (FBS) and 100 U/mL penicillin−streptomycin in a 5% CO2 environment. MCF-10A cells were cultured in DMEM/F-12 medium containing 5% horse serum, 100 U/mL penicillin, 100 mg/mL streptomycin, 100 ng/mL cholera toxin, 10 ng/mL epidermal growth factor, 0.5 mg/mL hydrocortisone, 10 mg/mL insulin, and 1% Lglutamine at 37 °C under an atmosphere of 5% CO2. After growing to 90% confluence, the cells were washed with PBS (0.145 M NaCl, 1.9 mM NaH2PO4, 8.1 mM K2HPO4, pH 7.4) and replaced the culture medium by 1 mL PBS. The cell number was estimated by a hemocytometer to be ∼1 × 105. Cellular Binding of the Aptamer−Ag-Au Nanostructures. Before the binding, the aptamer−Ag-Au nanostructures were labeled with Rh6G (Rhodamine 6G, a common Raman reporter) by incubating the nanostructures in an aqueous of Rh6G (10 μL of 0.1 μM Rh6G solution was added into 1 mL of aptamer−Ag-Au nanostructures solution) for 30 min. The Rh6G-labeled nanostructures were collected by centrifugation and washed thrice to remove the unadsorbed Rh6G molecules. The adsorption of Rh6G on the surface of the aptamer−Ag-Au nanostructures is stable as verified by fluorescence spectrum results (please refer to Figure S1 in the Supporting Information for details). After that, the Rh6G-labeled nanostructures were dispersed into PBS (pH 7.4) and incubated with the MCF-7 cells (∼1 × 105) for 30 min at 37 °C. The Rh6G-labeled nanostructures could bind onto the surface of cells via the specific interaction of S2.2 aptamer and the MUC1. The cells then were washed with PBS to remove nonbound nanostructures. The binding of the nanostructures onto the surfaces of the cells was verified by differential interference contrast (DIC), fluorescence, TEM images, and SERS spectra. The fluorescence images were captured under an AXIO microscope (AxioObserver A1, Carl Zeiss) using fluorescence mode with an excitation at 543 nm. The microscope was equipped with Epi− fluorescence and AxioCam MRc imaging system. DIC images were collected with transmitted light from the 543-nm

MUC1 mucin is a large transmembrane glycoprotein, whose expression increases at least 10-fold at the surface of MCF-7 cells in primary and metastatic breast cancers.5 This characteristic makes it an ideal target molecule for the detection of MCF-7 cells at the molecular level. Aptamers are small strands of DNA or RNA that could form unique three-dimensional (3D) structures that specifically combine with molecular targets with high affinity.6 Aptamers can function as effective molecular probes for the recognition of cancerous cells from complex living samples.7,8 Comparing to other targeting agents, aptamer possesses distinctive several advantages, such as ease of synthesis, stability in harsh biological environments, lack of immunogenicity, small size, and high affinity to targets.5,6 These properties make aptamers the most- valuable molecular probes for the sensitive recognition of specific cancer cells at very low concentrations. Among the published MUC1 aptamers, S2.2 is a 25-base oligonucleotide that binds to MUC1 protein with high affinity and specificity.9 This study constructed aptamer (S2.2)-guided Ag-Au nanostructures for specifically detecting the MCF-7 cells with the use of the high surface-enhanced Raman scattering (SERS) activity of the nanostructures. Raman spectroscopy is a powerful tool for cell detecting and imaging without suffering from rapid photobleaching and autofluorescence of biological samples.10−12 This technique can detect cells noninvasively and use near-infrared (NIR) excitation to ensure laser powers low enough for living cells to withstand.10 SERS enables the detection of very-lowconcentration analytes, even single molecules,13−15 ensuring highly sensitive detection of cancer cells even in the early stages of the disease. Ag-Au nanostructures are attractive SERS substrates, because of the synergism of these metals, the tunability of the plasmon resonance, and superior SERS activity.16−18 We prepared the aptamer−Ag-Au nanostructures by a simple photoreduction method with no use of any toxic chemical reductant, avoiding biological risks on the determination of living cells. At the same time, we herein also demonstrated that the aptamer−Ag-Au nanostructures are able to perform photothermal therapy of MCF-7 cells. The nanostructures have a high capability of adsorbing NIR irradiation and a high efficiency of delivering the generated heat to the cancer cell. Because the nanostructure binds to the MCF-7 cell surface via high specific interaction between the aptamer and MUC1, photothermal therapy can ensure the generation of high temperature at a desired cell, thus avoiding damaging healthy cells and destroying the surrounding normal tissue. Therefore, the synthesized nanostructures could offer a protocol to specifically recognize and sensitively detect MCF-7 cells, and would have great potential applications in the photothermal therapy of cancers.

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EXPERIMENTAL SECTION Synthesis and Characterization of Aptamer−Ag-Au Nanostructures. S2.2 aptamer (5′-GCA GTT GAT CCT TTG GAT ACC CTG G-3′, purchased from Sangon Bioengineering, Shanghai, China) was dissolved in phosphate buffer (PBS, pH 7.4) at a concentration of 300 ng/mL. Three hundred microliters (300 μL) of AgNO3 solution (1 mM) was added to the aptamer solution (100 μL). The molar ratio of Ag+ ions to aptamer (calculated by bases) was controlled to be 5. The solutions were mixed thoroughly and kept in darkness at 4 °C for 3 h. The photoreduction was performed directly in a microcuvette by placing the cuvette under 254 nm of ultraviolet 7693

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excitation. TEM images of the cells were recorded on a Hitachi Model 750 TEM microscope (Hitachi, Japan) operated at 80 kV. SERS Measurements. SERS spectra were recorded on a Labram HR 800 microspectrometer (Jobin−Yvon, France) with an excitation source of 785 nm. The objective used was an Olympus 50× long-working-distance lens (NA = 0.5) for both focusing the excitation beam on the sample and collecting the backscattering light onto an air-cooled charge-coupled device detector. The collection time of each SERS spectrum was 10 s over a spectral range from 400 cm−1 to 1800 cm−1. After binding with Rh6G-labeled aptamer−Ag-Au nanostructures, the cells were first rinsed with PBS thoroughly to remove the nonspecifically bound nanostructures and those nanostructures remaining in the medium, and then lysed with trypsin-EDTA solution. Finally, ∼10 μL of cells suspension was dropped onto the surface of freshly cleaved mica for SERS measurement. NIR Photothermal Therapy. For NIR photothermal therapy, we incubated MCF-7 cells (∼1 × 105) with aptamer−Ag-Au nanostructures (without Rh6G labeling) for 30 min. After that, the cells were repeatedly rinsed with PBS, and then exposed to irradiation with a wavelength of 808 nm at different power densities (0.06−0.25 W/cm2) for various time (5−60 min). Finally, the cells were treated with 0.4% Trypan Blue for 10 min to evaluate their viability. Living cells can get rid of Trypan Blue and keep themselves colorless but dead ones will collect the dye and turn blue; therefore, the cell viability can be observed from the color of the cells. The images of stained cells were collected in bright-field mode.

Figure 1. UV−vis absorption spectra showing the photoreduction synthesis of aptamer−Ag-Au nanostructures: (a) S2.2 aptamer in PBS (300 ng/mL); (b) aptamer−Ag+ mixture (the molar ratio of Ag+ ions to aptamer, calculated by bases, was 5); (c) the aptamer−Ag nanostructures; and (d) the aptamer−Ag-Au nanostructures (the molar ratio of AuCl4− ions to Ag+ ions was 4). Please note that the UV−vis absorption spectra, as depicted, have been reduced by a factor of 5 from the original signal for better observation of the intensity alteration and the position shift of the aptamer adsorption peak; therefore, the adsorption bands of Ag (∼420 nm) and Au (∼580 nm) seem to be weak and broad. Actually, the surface plasmon resonance band of Ag-Au nanostructures is strong enough to ensure high SERS activity. The inset shows the original intensity of the surface plasmon resonance band of the Ag-Au nanostructures.

These changes, which are related to the aptamer absorption, are attributed to UV-influenced supercoiling and aggregation/ cross-linking of aptamer strands due to trimeric complex formation.26 These results showed that Ag−Au nanostructures can be facilely grown on the aptamer chain by photoreduction. The aptamer played an active role in the formation of the nanostructures because irradiating an aqueous solution of AgNO3, NaAuCl4, or their mixtures without aptamer under the same conditions, did not result in the formation of Ag, Au, or Ag-Au nanostructures. UV irradiation may cause photodegradation of DNA and lead to break the DNA chain. To evaluate whether the undesired side effect occurred on the S2.2 aptamer under UV irradiation, we performed polyacrylamide gel electrophoresis (PAGE) experiment for the aptamer before and after UV irradiation (please refer to Supporting Information for the experimental details). We found the same length of the migration distance of aptamer prior to (see Lane 1 in Figure S2 in the Supporting Information) and upon UV irradiation (see Lane 2 in Figure S2 in the Supporting Information, at 254 nm with an exposure time of 60 min), which is indicative of no short DNA fragments being produced. This result suggested that the UV irradiation under our conditions did not cause the photodegradation of the aptamer; this was attributed to the presence of more metal cations than bases and the low power of UV irradiation that was adopted in our synthesis.21 TEM images were recorded for characterizing the morphologies of the synthesized nanostructures. Figure 2A shows a lower-magnification image that indicated a chainlike structure with a length of a few micrometers for the synthesized nanostructures. The nanostructures had been confined to grow along the chain of the aptamer, and very little background metal deposition was observed (Figure 2A). Figure 2B shows a higher-magnification image, illustrating that the chain of aptamer−Ag-Au had a branched structure, which was attributed to aggregation/cross-linking of aptamer on UV exposure,

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RESULTS AND DISCUSSION Synthesis of the Aptamer−Ag-Au Nanostructures. DNA can act as a template to guide the formation of metallic nanostructures as well as a stabilizer to prevent those formed nanostructures from precipitation.19−23 We synthesized the aptamer−Ag-Au nanostructures with photoreduction for specific detection and photothermal therapy of MCF-7 cells. The formation processes were monitored by UV absorption spectra. The aptamer (S2.2) showed a characteristic absorption peak at ∼267 nm (see curve a in Figure 1).19 Upon mixing with AgNO3 solution, the intensity had a significant increase (curve b in Figure 1), indicative of the formation of aptamer-Ag+ complex by embedding the Ag+ ions inside the DNA chain.19,20 Exposing the solution to 254-nm UV light resulted in the appearance of an absorption band centered at ∼420 nm (curve c in Figure 1). Such a band is attributed to the surface plasmon resonance of small Ag nanostructures produced under the photoreduction.24 Concomitantly, the aptamer absorption peak at 270 nm (has a 3-nm red shift, relative to that in curve a) showed a small decrease in intensity. These results indicated the formation of aptamer-Ag nanostructures. After the formation of aptamer−Ag nanostructures, the solution of AuCl4− was added into the system and photoreduced under 254-nm irradiation again. The solution turned blue upon photoreduction, with the appearance of an additional absorption band at ∼580 nm (curve d in Figure 1), which is characteristic of the surface plasmon resonance mode in Au nanoparticles22,25 and is indicative of the formation of Au nanostructures. However, the band related to the surface plasmon resonance of Ag disappeared, suggesting that the Ag nanostructures were covered by a layer of Au nanoparticles. Moreover, the intensity of aptamer absorption peak decreased with a red shift from 267 nm (curve a) to 287 nm (curve d). 7694

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Figure 2. (A−D) TEM images of the synthesized aptamer−Ag-Au nanostructures at different magnification. (E) HAADF-STEM image of the nanostructures, and the corresponding elemental mapping for (F) Ag and (G) Au. (H) Image obtained by overlapping panel F with panel G.

Figure 3. (A, B) DIC and (C, D) fluorescence images of the MCF-7 cells without (panels A and C) and with (panels B and D) binding of the Rh6Glabeled aptamer−Ag-Au nanostructures. (E) and (F) TEM images of the MCF-7 cells showing the binding of the Rh6G-labeled aptamer−Ag-Au nanostructures on the cell surface. Also shown are DIC images of (G) HepG2 and (H) MCF-10A after being incubated with the Rh6G-labeled aptamer−Ag-Au nanostructures. The inset in panel D is a high-magnification fluorescence image of the MCF-7 cells bound with Rh6G-labeled aptamer−Ag-Au nanostructures.

the nanoparticle (not shown here), which is same as the image shown in Figure 2H (superimposed images of the Au and Ag). The results also agreed well with those obtained from UV absorption. This structural feature benefits from the fact that the amount of AuCl4− precursor added into aptamer solution was much more than that of Ag+ ions, and a spontaneous galvanic replacement reaction between Ag nanoparticles and AuCl4− occurred.27−29 Such a unique feature of the synthesized nanostructures allows it to possess the good biocompatibility of Au and the high SERS activity of Ag nanostructures, and therefore can provide highly sensitive and specific detection of cancer cells. Specific Binding of the Aptamer−Ag-Au Nanostructures with Human Breast Cancer Cell. To demonstrate the possibility of the aptamer−Ag-Au nanostructures being used for specifically detecting MCF-7 cells, the nanostructures were labeled with Rh6G and then bound onto the surface of the cells

agreeing with the red shift of aptamer absorption peak on UV− vis spectra. The nanostructure had a core−shell structure, as revealed by the contrast between the shell and core (see Figures 2C and 2D). The size of the nanostructure was ∼100−120 nm (Figure 2C) with the diameter of the core being ∼80 nm and the thickness of the shell being ∼10 nm (Figure 2D), respectively. The high-angle annular dark-field scanning TEM (HAADF-STEM) image depicted in Figure 2E showed that the surface of the nanostructure was rough and comprised of several small particles ∼10 nm in size. To further obtain insight into the distribution of Ag and Au in the synthesized nanostructures, we carried out the elemental analysis with STEM (see Figures 2F−H). The results showed that Ag and Au were distributed throughout the core, and the shell was dominated with Au. Moreover, the line profile taken along the single Au−Ag nanoparticle revealed the distribution of Au and Ag throughout the core and the presence of Au at the edge of 7695

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nanostructures cannot attach to MCF-7 cells in the absence of S2.2 aptamer. The third control was made by directly incubating the MCF7 cells with Rh6G solution (free Rh6G molecules) for 30 min. After washing thrice, the cells exhibited a bright red fluorescence signal (Figure S5A in the Supporting Information). Moreover, the fluorescence was uniformly distributed on the cells, as viewed from the fluorescence image (Figure S5A in the Supporting Information) and the superimposed image of fluorescence with DIC images (Figure S5C in the Supporting Information), suggesting that the free Rh6G molecules were uniformly adsorbed on the surface of the cells without selectivity, because of the lack of a specific interaction, such as that between S2.2 and MUC1. On the contrary, the fluorescence signal of the cells is still not uniform, even after 2 h of incubation of the cells with Rh6G-labeled aptamer (S2.2)− Ag-Au nanostructures (see Figures S5D−F in the Supporting Information), suggesting that the nanostructures were specifically bound on the cells. Moreover, the images depicted in Figures S5D−F in the Supporting Information demonstrated that the adsorbed Rh6G molecules on the nanostructures were not desorbed; otherwise, the desorbed Rh6G would be adsorbed on the surface of the cells without specificity, causing the fluorescence signal on the surface of the cells to be distributed uniformly. We also incubated the HepG2 and MCF-10A cells with free Rh6G solution to check the adsorption selectivity of the free Rh6G molecules on these cells. The fluorescence images indicated that these cells showed uniformly distributed strong red fluorescence signal (see Figure S6 in the Supporting Information), demonstrating that no adsorption selectivity of the free Rh6G molecules occurred on the surface of the HepG2 and MCF-10A cells. The fourth control experiment was made by directly synthesizing the Ag−Au nanostructures (without aptamer), and labeling with Rh6G and incubating with MCF-7 cells. After washing thrice, the fluorescence images were recorded, and the results indicated that no fluorescence signal was detected (not shown here), suggesting that Ag-Au nanostructures grown in the absence of S2.2 aptamer could be easily washed away from the surface of the cells. These above results confirmed that the aptamer−Ag-Au nanostructures show high target specificity to MCF-7 cells, and can distinguish the cells from different cancer cell lines and normal breast cells; therefore, they can be used for the specific detection of this type of cancer cell. Moreover, these results indicated that the aptamer (S2.2) could still specifically recognize MUC1 on the surface of the cells, even after the growth of the Ag-Au nanostructures on its chain. SERS Detection of Human Breast Cancer Cell. Bimetallic alloys and core−shell nanomaterials, such as complexes of Au and Ag have been reported to have high SERS activity.16,20,33,34 Therefore, our synthesized aptamer− Ag-Au nanostructures are expected to be high SERS-active and can be used for specifically detecting MCF-7 cells. Rh6G was chosen as a reporter to study the SERS activity of the synthesized nanostructures, because of its well-established Raman spectral data and large Raman scattering cross section.35 The SERS spectrum of Rh6G on the aptamer−Ag-Au nanostructures showed the characteristic peaks of the C−C− C ring in-plane vibration (610 cm−1), C−H out-of-plane bending (771 cm−1), C−H in-plane bending (1127 and 1184 cm−1), N−H in-plane bending (1307 and 1575 cm−1), and C−

via the specific interaction of the MUC1 and the S2.2 aptamer. The binding of the nanostructures was first evidenced by the morphological changes of the cells. DIC image showed that the cell surface became much rougher after being incubated with the nanostructures (Figure 3B) in comparison with image of the cells before this incubation (Figure 3A), indicative of the binding of the nanostructures onto the cell surface. The fluorescence and TEM images also verified this binding process. As shown in Figure S3 in the Supporting Information, although the fluorescence of Rh6G was somewhat quenched when loaded on the aptamer−Ag-Au nanostructures, the remaining fluorescence signal was still strong enough to allow us to use fluorescence microscopy to check the binding features of the aptamer−Ag-Au nanostructures on the MCF-7 cells. The fluorescence image of the MCF-7 cells shows no signal, indicating no fluorescence emission of the cells (see Figure 3C). After the cells were incubated with Rh6G-labeled aptamer−Ag-Au nanostructures, a red fluorescence emitted from Rh6G was observed (see Figure 3D), indicating that the Rh6G-labeled nanostructures have bound onto the surface of the cells. The fluorescence image in high magnification (the inset in Figure 3D) showed that the fluorescence signal on the cell surface is uneven, suggesting that the distribution of MUC1 on the surface of MCF-7 cell is not uniform. TEM images showed several dark nanoclusters with a chainlike structure, which were the aptamer−Ag-Au nanostructures, which appeared on the surface of MCF-7 cells after being incubated with nanostructures (see Figures 3E and 3F). These results demonstrate that our synthesized aptamer−Ag-Au nanostructures can be targeted to MCF-7 cells. To further demonstrate that the Rh6G-labeled aptamer−AgAu nanostructures bind on the surface of the MCF-7 cells via the high specific interaction of the MUC1 and the S2.2 aptamer, four control experiments were performed. One control was made by incubating the Rh6G-labeled aptamer− Ag-Au nanostructures with HepG2 and MCF-10A cells. The fluorescence images indicated that no fluorescence signal was observed on these cells (not shown here). The DIC images also did not show any evidence of the nanostructures on the surface of the HepG2 and MCF-10A cells (see Figures 3G and 3H, respectively). These results suggested that the nanostructures could not be bound onto the surface of these cells, because both HepG2 and MCF-10A cells are MUC1-negative expressed. The second control was made by growing the Ag−Au nanostructures on a synthesized DNA (5′-GCA ATT GAT CCT ATG GAT ACC ATG G-3′, having three mismatched bases, in comparison with S2.2), on sgc8 apatmer (5′-ATC TAA CTG CTG CGC CGC CGG GAA AAT ACT GTA CGG TTA GA-3′, specific for CEM cells30,31), and on TDO5 aptamer (5′-AAC ACC GGG AGG ATA GTT CGG TGG CTG TTC AGG GTC TCC TCC CGG TG-3′, specific for Ramos cells32) chain, respectively, and then labeling with Rh6G and incubating with MCF-7 cells. After washing thrice, the DIC and fluorescence images were recorded. DIC images as depicted in Figures S4A−C in the Supporting Information showed that the morphologies of the cells (in particular, the surface roughness) remained almost unchanged, compared with that of the cells before incubation (as depicted in Figure 3A). The fluorescence images depicted in Figure S4D-F in the Supporting Information also showed no detectable fluorescence signal after the cells were incubated with those Rh6G-labeled nanostructures. These results indicated that the Ag−Au 7696

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C stretching vibration mode (1362, 1510, and 1650 cm−1) (see Figure S7a in the Supporting Information), as reported previously.4,36,37 The SERS signals were much higher than those recorded at aptamer-Ag (Figure S7b in the Supporting Information) and aptamer-Au nanostructures (Figure S7c in the Supporting Information), and the analytical enhancement factor (AEF) was calculated to be ∼1.5 × 106 for the aptamer− Au-Ag nanostructures with use of eq 1,4,17,38 signifying the superior SERS active substrate of the aptamer−Au-Ag nanostructures (noted that the value of AEF was calculated on the basis of the SERS of Rh6G on the aptamer−Ag-Au nanostructures in solution, not bound to the cell). ⎛I ⎞ ⎛ C ⎞ AEF = ⎜ SERS ⎟ × ⎜ RS ⎟ ⎝ IRS ⎠ ⎝ CSERS ⎠

These results suggested that a new assay has been developed for detecting the MCF-7 cells, using the high SERS activity of the aptamer−Ag-Au nanostructures. Because the SERS technique can provide ultrasensitive detection, the assay can be used for highly specific recognition and the highly sensitive detection of MCF-7 cells. NIR Photothermal Therapy of Human Breast Cancer Cells. Photothermal therapy is currently considered to be a relatively noninvasive and benign alternative for cancer treatment that can lead to photoablation of the cancer cells and subsequent cell death.4,39−44 In photothermal therapy, the absorbed light is converted to heat and transferred to the cell environment, generating localized hyperthermia and destroying malignant cells. After successfully demonstrating the specific detection of MCF-7 cells with the synthesized aptamer−Ag-Au nanostructures, we performed NIR irradiation experiments to determine whether the nanostructures can be used for photothermal therapy of the cancer cells (please note that nanostructures with no Rh6G labeling were used in the photothermal experiments). We incubated the MCF-7 cells with the aptamer−Ag-Au nanostructures and irradiated them with a wavelength of 808 nm. The cell viability was evaluated directly from optical observations through staining Trypan Blue. After exposure to a laser power density of 0.06 W/cm2 for 60 min, the cells were still living well (see Figure 5A). However, when the irradiation power density increased to 0.13 W/cm2, several blue dots appeared, which signified cell death. (See Figure 5B; please note that the blue dots are the nucleus of stained dead cells, not the aggregates of the Ag−Au nanostructures. To verify that the large blue dots are the nucleus of stained dead cells, we performed a control experiment by treating the dead cells (without binding the Ag−Au nanostructure) with 0.4% Trypan Blue for 10 min, and then optical microscopy images were recorded. The images (not shown here) indicated that the dead cells had similar blue dots as those in Figures 5B−D), and the number of death cells increased as the irradiation power density increased (see Figures 5B−D). When the power reached 0.25 W/cm2, almost all of the cells were stained with Trypan Blue. In comparison, we observed no destruction for the cells without incubating them with the nanostructures, even at the highest power density of 0.25 W/cm2 (not shown here). Therefore, it can be concluded that the cell deaths shown in Figures 5B−D are a consequence of the local heating generated from the Ag-Au nanostructures under NIR irradiation. It is worth noting that the irradiation power density (0.25 W/cm2) needed to achieve substantial cells death is much lower than those reported previously for reduced graphene oxide (0.6 W/cm2),39 chitosan-Au nanorods (3 W/cm2),45 Au nanorods (8.5 W/ cm2),46 and popocorn-shaped Au nanoparticles (12.5 W/cm2),4 which is indicative of the high efficiency of the synthesized nanostructures in photothermal therapy. Further evidence of the increased cell mortality following irradiation was provided by MTT assay,47,48 which involves a colorimetric assay for assessing the viability of cells (please refer to the Supporting Information for experimental details). The results depicted in Figure 5E showed that only a limited decrease in viability was observed when the cells were irradiated at 0.06 W/cm2. However, the loss of the cell viability is significant upon the increase in the irradiation power densities. The cell viability decreased to ∼75%, 46%, and 3% at the power densities of 0.13, 0.19, and 0.25 W/cm2, respectively, showing an irradiation-power-density-dependent manner, agreeing well

(1)

where ISERS is the intensity of a 1362 cm−1 vibration mode in SERS of Rh6G at the aptamer−Au-Ag nanostructures and IRS is the Raman intensity of the same mode for Rh6G in the bulk solution (the Raman spectrum of Rh6G in solution is depicted in Figure S8 in the Supporting Information). CSERS and CRS are the concentration of Rh6G used for SERS and normal Raman signal recording, respectively. The high Raman activity of the synthesized aptamer−Ag-Au nanostructures is useful for the specific detection of MCF-7 cells. The detection was based on the SERS signal of the Rh6G reporter on the nanostructures. After binding with the Rh6Glabeled aptamer−Ag-Au nanostructures, the SERS spectrum of the MCF-7 cells displayed the characteristic peaks of Rh6G (Figure 4a). However, the SERS spectrum of the cells without

Figure 4. SERS spectra of the MCF-7 cells with and without binding of the Rh6G-labeled aptamer−Ag-Au nanostructures (spectra a and b, respectively). SERS spectra of the HepG2 and MCF-10A (spectra c and d, respectively), after being incubated with the Rh6G-labeled aptamer−Ag-Au nanostructures, also are shown.

binding with the nanostructures did not show any signal (Figure 4b), indicating that the nanostructures can be used to detect MCF-7 cells, based on their high SERS activity. The selectivity of this detection is demonstrated by use of the HepG2 and MCF-10A cells as controls. After being incubated with the Rh6G-labeled aptamer−Ag-Au nanostructures, both the HepG2 and MCF-10A cells did not show any SERS signal of the reporter (see spectra c and d in Figure 4) because the nanostructures cannot be bound to these MUC1-negative expressed cells.5 These results agreed well with those obtained from fluorescence and DIC images (see Figure 3G and 3H). 7697

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Figure 5. Microscopic images of MCF-7 cells after being incubated with the aptamer−Ag-Au nanostructures and irradiated for 60 min at power densities of (A) 0.06, (B) 0.13, (C) 0.19, and (D) 0.25 W/cm2, respectively. The images were recorded after staining with Trypan Blue. (Also shown are MTT assays showing the dependence of the cell viability of MCF-7 cells on (E) the irradiation power density and (F) the irradiation time. (G) Effects of the photothermal therapy on the amount of LDH release for the MCF-7 and MCF-10A cells. (H) Microscopic image of MCF-10A cells after being incubated with the synthesized nanostructures and irradiated for 60 min at 0.25 W/cm2.

application in clinical therapy. It is noteworthy that the clinical therapeutic efficiency can be improved significantly by the combination of localized hyperthermia treatment and chemotherapy.

with the optical microscopy observations. We also performed an irradiation time-dependent MTT assay to understand whether the efficiency of the photothermal therapy depends on the irradiation time (Figure 5F). The cells viability was ∼92% after 5 min of irradiation, and decreased rapidly as the irradiation time increased. Almost all of the cancer cells were dead after 60 min of irradiation, suggesting that 60 min of irradiation is sufficient for therapy of the cells, using the synthesized nanostructures. To evaluate the safety of photothermal therapy based on our aptamer−Ag-Au nanostructures, we performed the control experiments using MCF-10A cells following the same treatment regime as the MCF-7 cells. Optical observations indicated that the MCF-10A cells were still living (Figure 5H) well after irradiation for 60 min, even at a high power density of 0.25 mW/cm2. A CytoTox-One assay was also performed for the MCF-7 and MCF-10A cells to assess the damage to the cellular membrane following photothermal therapy (please refer to the Supporting Information for experimental details).49,50 This assay is a fluorescent measure of the release of lactate dehydrogenase (LDH), which is a cell membrane damage marker, from cells with a damaged membrane. A high level of membrane damage was observed for the MCF-7 cells following the photothermal therapy, as evidenced by the increase in LDH (see Figure 5G). However, the membrane permeability was not compromised in the MCF-10A cells after the photothermal therapy. These results, combined with optical microscopy observations, clearly indicate that the aptamer−Ag-Au nanostructures can effectively avoid killing normal cells in photothermal therapy, representing a high specificity. This is because the nanostructures cannot be targeted to the MCF-10A cells via the interaction of MUC1 and the aptamer, because the MCF-10A cells are MUC1-negative expressed. Therefore, highly specific detection and photothermal therapy based on our aptamer−Ag-Au nanostructures may be of great scientific significance in the cancer research area and have a potential

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CONCLUSIONS In summary, the aptamer−Ag-Au nanostructures have been synthesized by photoreduction for specifically detecting MCF-7 cells at a molecular level. With the use of a Raman reporter of Rh6G to enhance the sensitivity, the nanostructures can be used for the highly specific detection of a single MCF-7 cell, as well as discrimination of MCF-7 cells from normal breast cells and different cancer cell lines. In addition, the nanostructures have a high capability of adsorbing near-infrared (NIR) irradiation and are able to perform photothermal therapy of MCF-7 cells at a very low irradiation power density without causing the destruction of healthy cells and the surrounding normal tissue. The synthesized nanostructures could offer a protocol to specifically recognize and sensitively detect the cancer cells, and would have a great potential application in the photothermal therapy of cancers.

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ASSOCIATED CONTENT

S Supporting Information *

The experimental details on the electrophoresis, MTT assay, and CytoTox-One homogeneous membrane integrity assay, and eight figures. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest. 7698

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ACKNOWLEDGMENTS This work is supported by the National Natural Science Foundation of China (Nos. 21273117 and 21175067), the Research Fund for the Doctoral Program of Higher Education of China (No. 20103207110004), the Natural Science Foundation of Jiangsu Province (No. BK2011779), the Foundation of the Jiangsu Education Committee (Nos. 09KJA150001 and 10KJB150009), the Program for Outstanding Innovation Research Team of Universities in Jiangsu Province, and the Priority Academic Program Development of Jiangsu Higher Education Institutions.

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dx.doi.org/10.1021/ac3015164 | Anal. Chem. 2012, 84, 7692−7699