Using Aptamer-Conjugated Fluorescence Resonance Energy Transfer

Jul 2, 2009 - Lin-Chen Ho , Wei-Cheng Wu , Chang-Yu Chang , Hao-Hsuan Hsieh , Ching-Hsiao ... Weihong Tan , Michael J. Donovan , and Jianhui Jiang...
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Anal. Chem. 2009, 81, 7009–7014

Using Aptamer-Conjugated Fluorescence Resonance Energy Transfer Nanoparticles for Multiplexed Cancer Cell Monitoring Xiaolan Chen,†,‡ M.-Carmen Este´vez,† Zhi Zhu,† Yu-Fen Huang,† Yan Chen,† Lin Wang,§ and Weihong Tan*,† Center for Research at the Bio/Nano Interface, Department of Chemistry and Department of Physiology and Functional Genomics, Shands Cancer Center, UF Genetics Institute and McKnight Brain Institute, University of Florida, Gainesville, Florida 32611-7200, Department of Chemistry, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, 361005, People’s Republic of China, and Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285 To facilitate the selection of effective therapeutic pathways and improve clinical outcomes, sensitive and simultaneous diagnosis of multiple trace biomarkers or cancer cells from complex living samples is particularly critical in the early stages of tumor development. To achieve this, we have combined the selectivity and affinity of aptamers with the spectroscopic advantages of fluorescence resonance energy transfer (FRET) nanoparticles (NPs). This has produced an aptamer-conjugated FRET NP assay that performs simultaneous multiplexed monitoring of cancer cells with the desired degree of sensitivity and selectivity. First, by changing the doping ratio of three different dyes, the FRET-mediated emission signatures could be tuned such that the nanoparticles would exhibit multiple colors upon excitation with a single wavelength. These FRET nanoparticles were then modified by a few aptamers specific for different cancer cell lines, in this case, T-cell leukemia and B-cell lymphoma. As a result, simultaneous and sensitive detection of multiple cancer cell targets was achieved. Therefore, our aptamer-conjugated FRET NPs are highly promising for potential applications in the sensitive monitoring of multiple cancer cells for biomedical research and medical diagnostics. How to recognize multiple trace biomarkers or cancer cells from complex living samples is a critical issue for the early diagnosis of cancer.1,2 Simultaneous monitoring of different types of cancer cells in a single assay would require the recognition of specific receptors on the cell membrane surface. However, it is precisely the lack of specific molecular probes for cancer biomarkers that, in part, hinders greater progress in this area. To * To whom correspondence should be addressed. Tel./Fax: (+1) 352-8462410. E-mail: [email protected]. † Center for Research at the Bio/Nano Interface, Department of Chemistry and Department of Physiology and Functional Genomics, Shands Cancer Center, UF Genetics Institute and McKnight Brain Institute, University of Florida. ‡ Department of Chemistry, College of Chemistry and Chemical Engineering, Xiamen University. § Lilly Research Laboratories, Eli Lilly and Company. (1) Lee, H. J.; Wark, A. W.; Corn, R. M. Analyst 2008, 133, 975–983. (2) Fredriksson, S.; Horecka, J.; Brustugun, O. T.; Schlingemann, J.; Koong, A. C.; Tibshirani, R.; Davis, R. W. Clin. Chem. 2008, 54, 582–589. 10.1021/ac9011073 CCC: $40.75  2009 American Chemical Society Published on Web 07/02/2009

address this limitation, aptamers, which are single-stranded oligonucleotides, represent an attractive alternative to antibodies as specific ligands to recognize multiple trace biomarkers or cancer cells from complex living samples. Using the SELEX process (systematic evolution of ligands by exponential enrichment), aptamers are selected from a DNA or RNA pool by repetitive binding of target molecules.3,4 In this way, aptamers can be selected for a wide variety of targets, from small molecules to proteins,5-7 showing affinity and selectivity that is comparable to the commonly used antibodies.8,9 In addition, aptamers also possess some additional advantages, such as their relatively small size, lack of immunogenicity, as well as the ease of synthesis and modification. All these factors lead to their increasing use in bioanalysis, chemical biology, biomedicine, and biotechnology.10 When applying whole living cells as a target, a panel of aptamers can be generated for the recognition of unique molecular signatures on the surface of cells through an enhanced cell-based SELEX process.11,12 Using tumor cancer cells as targets, specific aptamers can be selected with the ability to distinguish cancer cells from normal cells and, moreover, to differentiate a particular tumor type among various strains.13-15 These properties, combined with their high affinity, make aptamers effective for use in (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13)

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Tuerk, C.; Gold, L. Science 1990, 249, 505–510. Ellington, A. D.; Szostak, J. W. Nature 1990, 346, 818–822. Liu, J. W.; Lu, Y. J. Am. Chem. Soc. 2004, 126, 12298–12305. Famulok, M.; Mayer, G. Curr. Top. Microbiol. Immunol. 1999, 243, 123– 136. Floch, F. L.; Ho, H. A.; Leclerc, M. Anal. Chem. 2006, 78, 4727–4731. Brody, E. N.; Gold, L. J. Biotechnol. 2000, 74, 5–13. Jayasena, S. D. Clin. Chem. 1999, 45, 1628–1650. Navani, N. K.; Li, Y. F. Curr. Opin. Chem. Biol. 2006, 10, 272–281. Morris, K. N.; Jensen, K. B.; Julin, C. M.; Weil, M.; Gold, L. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 2902–2907. Daniels, D. A.; Chen, H.; Hicke, B. J.; Swiderek, K. M.; Gold, L. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 15416–15421. Shangguan, D. H.; Li, Y.; Tang, Z. W.; Cao, Z. H. C.; Chen, H. W.; Mallikaratchy, P.; Sefah, K.; Yang, C. Y. J.; Tan, W. H. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 11838–11843. Sefah, K.; Tang, Z.; Shangguan, D.; Chen, H.; Lopez-Colon, D.; Li, Y.; Parekh, P.; Martin, J.; Meng, L.; Phillips, J. A.; Kim, Y.; Tan, W. Leukemia 2009, 23, 235–244. Tang, Z. W.; Shangguan, D. H.; Wang, K. M.; Shi, H.; Sefah, K.; Mallikaratchy, P.; Chen, H. W.; Li, Y.; Tan, W. H. Anal. Chem. 2007, 79, 4900–4907.

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diagnostic assays, including the simultaneous monitoring of different cell types, or targeted therapeutic applications. At the same time, the rapidly evolving field of nanoscience and nanotechnology has brought a variety of novel approaches to the diagnosis and therapy of diseases based on the use of NPs with different chemical and physical properties. Among these, fluorescent silica NPs are one of the most widely developed and used NPs.16-23 They possess some key advantages over conventional organic dyes. For example, they have a much stronger optical signal than a single-dye molecule, and they are more stable against photobleaching. Moreover, the surface of silica NPs provides a robust and stable shell, usually with a versatile composition that allows easy manipulation and feasible functionalization, either through physical adsorption or covalent attachment. The combination of fluorescent silica NPs and aptamers has already been used in the detection of cancer cells,24,25 displaying their potential in tumor diagnosis. However, when different single-dye-doped fluorescent silica NPs are applied for multiplexed analysis, there is often a need for multiple laser lights to excite them separately.26 This can require complex operating procedures and expensive instruments. Using a single excitation, on the other hand, could minimize the complexity of fluorescent detection, thereby considerably simplifying the instrumentation requirements, and meanwhile allowing a simultaneous excitation of all fluorophores involved in the assay. Recently, fluorescent silica NPs doped with three fluorescence resonance energy transfer (FRET) dyes have been prepared and applied in the simultaneous and sensitive detection of multiple targets.27,28 These dyes possess appropriately overlapped excitation and emission spectra, so that efficient energy transfer among them can occur. Thus, using a single wavelength excitation, but varying the ratios of three dyes coencapsulated into the silica NPs, different emission signatures can be obtained. This type of fluorescent NPs has already been conjugated with antibodies for the simultaneous detection of bacteria.28 In this work, we further extend the use of FRET NPs for multiplexed cancer cell monitoring by exploiting the unique spectroscopic advantages of FRET NPs and the high specificity and affinity of aptamers. Aptamers were selected from three different cancer cell lines, which were used as the target cells. Fluorescence imaging and flow cytometry were used to confirm (16) Wang, L.; Wang, K. M.; Santra, S.; Zhao, X. J.; Hilliard, L. R.; Smith, J.; Wu, Y. R.; Tan, W. H. Anal. Chem. 2006, 78, 648–654. (17) Ye, Z. Q.; Tan, M. Q.; Wang, G. L.; Yuan, J. L. Anal. Chem. 2004, 76, 513–518. (18) Xia, X. H.; Xu, Y.; Zhao, X. L.; Li, Q. G. Clin. Chem. 2009, 55, 179–182. (19) Chen, X. L.; Zou, J. L.; Zhao, T. T.; Li, Z. B. J. Fluor. 2007, 17, 235–241. (20) Nakamura, M.; Shono, M.; Ishimura, K. Anal. Chem. 2007, 79, 6507–6514. (21) Wu, H.; Huo, Q. S.; Varnum, S.; Wang, J.; Liu, G. D.; Nie, Z. M.; Liu, J.; Lin, Y. H. Analyst 2008, 133, 1550–1555. (22) Ha, S. W.; Camalier, C. E.; Beck, G. R., Jr.; Lee, J. K. Chem. Commun. 2009, 2881–2883. (23) Burns, A. A.; Vider, J.; Ow, H.; Herz, E.; Penate-Medina, O.; Baumgart, M.; Larson, S. M.; Wiesner, U.; Bradbury, M. Nano Lett. 2009, 9, 442–448. (24) Herr, J. K.; Smith, J. E.; Medley, C. D.; Shangguan, D. H.; Tan, W. H. Anal. Chem. 2006, 78, 2918–2924. (25) Smith, J. E.; Medley, C. D.; Tang, Z. W.; Shangguan, D. H.; Lofton, C.; Tan, W. H. Anal. Chem. 2007, 79, 3075–3082. (26) Wang, L.; O’Donogbue, M. B.; Tan, W. H. Nanomedicine 2006, 1, 413– 426. (27) Wang, L.; Tan, W. H. Nano Lett. 2006, 6, 84–88. (28) Wang, L.; Zhao, W. J.; O’Donogbue, M. B.; Tan, W. H. Bioconjugate Chem. 2007, 18, 297–301.

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the selectivity of the assay. Our results indicate that these FRET NP-aptamer conjugates can be used as a highly sensitive and selective platform for sensing multiple cancer cells simultaneously and can therefore provide a new method of large-scale screening for particular diseases. EXPERIMENTAL SECTION Chemicals. Amine-reactive dyessFAM-SE (5-carboxyfluorescein succinimidyl ester), R6G-SE (5-carboxyrhodamine 6G, succinimidyl ester), and ROX-SE (6-carboxy-X-rhodamine, succinimidyl ester)swere obtained from Invitrogen (Carlsbad, CA). NHSPEG5000-biotin was ordered from Jenkem Technology (Allen, TX). Neutravidin was obtained from Pierce Biotechnology, Inc. (Rockford, IL), and ammonium hydroxide (28.0%-30.0%) was purchased from Fisher Scientific Co. TEOS (tetraethyl orthosilicate) and APTES [(3-aminopropyl)triethoxysilane)] were purchased from Aldrich Chemical Co. (St. Louis, MO). THPMP [(3-Trihydroxysilyl) propyl methyl-phosphonate] was purchased from Gelest, Inc. (Morrisville, PA). All other chemicals were of analytical reagent grade. Distilled deionized water (Easy Pure LF) was used for the preparation of all aqueous solutions. Cell Lines and Buffers. Ramos cells (CRL-1596, B lymphocyte, human Burkitt’s lymphoma), CCRF-CEM cells (CCL-119, T lymphoblast, human acute lymphoblastic leukemia), and Toledo cells (CRL-2631, B lymphocyte, human diffuse large cell lymphoma) were obtained from the American Type Culture Association. The cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) (heat inactivated, GIBCO) and 100 IU/mL penicillin-streptomycin (Cellgro). The washing buffer contained 4.5 g/L glucose and 5 mM MgCl2 in Dulbecco’s PBS (Sigma). Binding buffer was prepared by adding yeast tRNA (0.1 mg/mL) (Sigma) and BSA (1 mg/mL) (Fisher) into the washing buffer to reduce background binding. The cell density was determined using a hemocytometer, and it was calculated prior to any experiments. Cells were dispersed in washing buffer, centrifuged at 970 rpm for 3 min, and redispersed in binding buffer for incubation with NPs. During all experiments, the cells were kept in an ice bath at 4 °C. Fluorescent NPs Preparation. The single-dye-, dual-dye-, and triple-dye-doped silica NPs were prepared according to the method that we have developed.27 Briefly, amine-reactive dye molecules (FAM-SE, R6G-SE, and ROX-SE) were first individually dissolved in 0.5 mL of anhydrous DMSO (dimethylsulfoxide), and APTES was then added to each solution at a dye:APTES molar ratio of 1:2. The suspension was kept under darkness and stirred for 12 h. The FAM-APTES, R6G-APTES, and ROX-APTES conjugates mixed at desired ratios (i.e., FAM: R6G (0.5:2) and FAM:R6G: ROX (0.5:1:4)) were then added to a clean glass reaction vessel that contained 16.75 mL of ethanol and 1.275 mL of ammonium hydroxide. The mixture was allowed to react for 24 h. TEOS (0.71 mL) was then added and continued to react for another 24 h. The final NPs were recovered by centrifugation at 14 000 rpm for 15 min, and the particles were washed continuously with ethanol and water several times to remove any free dye or unreacted materials. The size of the NPs was determined by TEM analysis (Hitachi H7100, Tokyo, Japan). The NPs appeared to be highly monodisperse with average diameters for NP(FAM), NP(FAM-R6G), and NP(FAM-R6G-ROX) of 63 ± 4.0 nm, 64 ± 5.0 nm, and 60 ± 6.0 nm, respectively. The fluorescence spectra of the three different

NPs were recorded on a Fluorolog TAU-3 spectrofluorometer (Jobin Yvon-Spex, Instruments S.A., Inc., Edison, NJ). Synthesis of DNA Aptamers. Three different aptamers with the equilibrium dissociation constants in the nanomolar to subnanomolar range previously selected for three different cancer cell lines were used in the experiment: sgc8 aptamer13,14 (Kd ) 0.80 nM), 5′-ATC TAA CTG CTG CGC CGC CGG GAA AAT ACT GTA CGG TTA GA-3′, specific for CEM cells; TDO5 aptamer15 (Kd ) 74.7 nM), 5′-AAC ACC GGG AGG ATA GTT CGG TGG CTG TTC AGG GTC TCC TCC CGG TG-3′, specific for Ramos cells; and T1 aptamer14 (also donated as sgd5, Kd ) 70.8 nm), 5′-ATA CCA GCT TAT TCA ATT ATC GTG GGT CAC AGC AGC GGT TGT GAG GAA GAA AGG CGG ATA ACA GAT AAT AAG ATA GTA AGT GCA ATC T-3′, specific for Toledo cells, respectively. The biotinylated versions of the aptamer sequences at the 3′-end were synthesized in-house, using 3′-Biotin-CPG, and all of them had an additional poly T linker (10 T bases, PolyT10) between the biotin and the specific sequence. An ABI3400 DNA/RNA synthesizer (Applied Biosystems, Foster City, CA) was used for the synthesis of all DNA sequences. A ProStar HPLC (Varian, Walnut Creek, CA) with a C18 column (Econosil, 5 µm, 250 × 4.6 mm) from Alltech (Deerfield, IL) was used to purify all fabricated DNA. All oligonucleotides were synthesized by solid-state phosphoramidite chemistry at a 1-µmol scale. The completed sequences were then deprotected in AMA (ammonium hydroxide:40% aqueous methylamine, 1:1) at 65 °C overnight and further purified twice with reverse-phase high-pressure liquid chromatography (HPLC) on a C-18 column. A Cary Bio-300 UV spectrometer (Varian, Walnut Creek, CA) was used to measure the absorbance to quantify the manufactured sequences. Preparation of Nanoparticle-Aptamer Conjugates. The biotinylated aptamers were immobilized onto the surface of neutravidin-coated fluorescent NPs.29 Briefly, the NPs were initially modified with amino groups, which were coupled to a PEG linker (MW ) 5000) with an NHS group at one end and a biotin group at the other end. Subsequently, neutravidin was immobilized onto the NP surface through biotin-avidin interaction. The biotinylated aptamer was further incubated with the neutravidincoated NPs (1 mg/mL), with an aptamer:NP molar ratio of 1000: 1. The solution was allowed to gently shake overnight at 4 °C. The conjugates were washed three times with phosphate buffer and finally reconstituted in binding buffer and stored at 4 °C until use. Flow Cytometry Analyses. To demonstrate the targeting capabilities of NP-conjugated aptamers toward specific cells, fluorescence measurements were conducted using a FACScan cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA). Typically, the binding experiments were performed using the following procedure. Approximately 5 × 105 of pure cells, or a mixture of the different cell lines (the total amount of cells being 5 × 105), were obtained from the culture media and placed in individual test tubes; 10 µL of the NP-aptamer solution (1 mg/mL) were added to the cell suspension, and the mixture was incubated on ice for 20 min. After incubation, the cells were washed twice by centrifugation with washing buffer (0.8 (29) Estevez, M.-C.; O’Donoghue, M. B.; Chen, X. L.; Tan, W. H. Nano Res. 2009, 2, 448–461.

mL) and finally resuspended in binding buffer (0.2 mL). The mean fluorescence was determined by counting 30 000 events. Neutravidin-coated NPs without aptamer on the surface were used as a negative control. Depending on the NPs used the signal was monitored in channel 1 for NP(FAM), channel 2 for NP(FAM-R6G), or channel 3 for NP(FAM-R6G-ROX). Cell Imaging. Confocal images of the previously described cell-NP suspensions were taken by depositing 10 µL of the suspension onto a thin glass slide placed above a 20 × 2 objective on the confocal microscope. Fluorescence imaging was conducted on a confocal microscope setup consisting of an Olympus IX-81 inverted microscope with an Olympus Fluoview 500 confocal scanning system and three lasers, a tunable argon-ion laser (458, 488, 514 nm), a green HeNe laser (543 nm), and a red HeNe laser (633 nm) with three separate photomultiplier tubes (PMTs) for detection. The fluorescent NPs were excited with the 488 nm line of the argon-ion laser, and emission was detected using a 505-525 nm bandpass filter for FAM signal, a 535-565 nm bandpass filter for the R6G signal, and a 610-nm long-pass filter for the ROX signal. RESULTS AND DISCUSSION Preparation and Characterization of Fluorescent Nanoparticles. Fluorescent silica NPs that incorporated different dyes and had an average particle size of ∼60 nm were prepared as recently described.27 The three dyes (FAM, R6G, and ROX) were chosen to allow an efficient-energy transfer between them. Thus, in this case, three different NPs were prepared: one contained a single dye, NP (FAM); another one contained two dyes, NP (FAMR6G); and, finally, one had three dyes, NP (FAM-R6G-ROX). In the dual-dye-doped NPs, FAM (λex ) 488 nm, λem ) 520 nm) was used as a donor for R6G (λex ) 528 nm, λem ) 550 nm), whereas in the triple-dye-doped NPs, FAM can act as a common donor for R6G and ROX (λex ) 580 nm, λem ) 605 nm), and, at the same time, R6G acted as both an acceptor for FAM and a donor for ROX. By changing the ratios of FAM:R6G, or FAM: R6G:ROX, different emission signature properties can be obtained under a single wavelength excitation (λex ) 488 nm). The final ratios used for the preparation of the three NPs was FAM:R6G:ROX 1:0:0 for NP(FAM), FAM:R6G:ROX 0.5:2:0 for NP (FAM-R6G) and FAM:R6G:ROX 0.5:1:4 for NP(FAM-R6GROX). As shown in Figure 1, three different maximum emissions of 523, 561, and 614 nm for the three NPs were obtained at a single wavelength excitation of 488 nm. Generally, the number of dyes trapped inside the NPs can be determined by comparing the fluorescence signal of the NP solution with a calibration curve of the pure dye under the same conditions. For example, for NP(FAM), the average fluorescence of the NPs was equivalent to ∼3200 dyes/NP. However, for NP (FAM-R6G) and NP (FAMR6G-ROX), it is difficult to address the accurate dye numbers, because the fluorescence intensity that corresponds to FAM and R6G is suppressed by energy transfer. The three NPs were further surface-modified with amino groups to allow for the incorporation of a polyethylene glycol (PEG) linker (MW ) 5000) with an NHS-reactive group at one end and a biotin group at the other. Next, a layer of neutravidin was added to the PEG layer. Recently, we studied the surface modification of silica NPs to decrease their inherent nonspecific adsorption onto the cell surface,29 and we observed that the Analytical Chemistry, Vol. 81, No. 16, August 15, 2009

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Figure 1. Fluorescence emission spectra of NP(FAM) (black), NP(FAM-R6G) (green), and NP(FAM-R6G-ROX) (red) at a concentration of 2.5 mg/mL. The maximum emission is 523 nm for NP(FAM), 561 nm for NP(FAM-R6G), and 614 nm for NP(FAM-R6G-ROX).

introduction of a PEG linker layer onto the silica layer, followed by an extra layer of neutravidin, provided an efficient coating that could avoid nonspecific interactions with cells and meanwhile conveniently immobilize the aptamers of interest by just first introducing a biotin group in the aptamer sequence. NPs-Cancer Cells Binding Specificity. Three different cancer cell lines, against which several specific aptamers had been previously selected, were chosen to perform the cell-specificity and multiplexing experiments. The biotinylated aptamers were coupled to the neutravidin-coated NPs, as previously described.29 In multiplexing assays, the specificity of the probes in binding the corresponding target is crucial for accurate results. The aptamers selected had already been proven to be specific for the corresponding cell type and had affinity constants in the nanomolar range. Thus, sgc8 aptamer was selected for CEM cells (which are human acute lymphoblastic leukemia cells),13,14 TDO5 aptamer was selected against Ramos cells (which are human Burkitt’s lymphoma cells),15 and T1 aptamer was selected for Toledo cells (which represent a type of human diffuse large cell lymphoma).14 T1 aptamer was coupled to NP(FAM), sgc8 aptamer to NP(FAM-R6G), and TDO5 aptamer to NP(FAM-R6G-ROX). Here, the specificity of the FRET NP-aptamer probes was determined by flow cytometry and confocal microscopy. Each conjugatesNP(FAM)-T1, NP(FAM-R6G)-sgc8, and NP(FAMR6G-ROX)-TDO5, respectivelyswas initially incubated with pure cells, and the binding was monitored by flow cytometry. As shown in Figure 2, the three NP-aptamer conjugates showed high specificity for the target cells (green line), whereas the other two cell lines showed little or no fluorescence, indicating that the binding capability of the aptamer probes is maintained well after the conjugation with NPs. Moreover, the background signals that originate from the possible nonspecific interaction of the plain neutravidin-coated NP was irrelevant for the three different cells (denoted by the red line in Figure 2). Similarly, confocal images showing individual incubation of each conjugate with pure cells also confirmed the specificity of the NP-aptamer conjugates for the corresponding cells (see Figure 3). As shown in Figure 3, the resulting colors, after merging the three different emission channels used for monitoring the three NPs, are clearly differentiated, displaying blue for NP(FAM) with Toledo cells, light green 7012

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Figure 2. Flow cytometry histograms of the three NP-aptamer conjugates with the three different cells (Toledo, CEM, and Ramos). (A) NP(FAM)-T1; (B) NP(FAM-R6G)-sgc8, and (C) NP (FAM-R6GROX)-TDO5. The black line corresponds to pure cells, the red line corresponds to NPs without aptamer, and the green line corresponds to NP-aptamer conjugates.

Figure 3. Confocal microscopy images of individual NP-aptamer conjugates with the three different cells (Toledo, CEM, and Ramos): (A) NP (FAM)-T1, (B) NP (FAM-R6G)-sgc8, and (C) NP (FAM-R6GROX)-TDO5.

for NP(FAM-R6G) with CEM cells, and purple red for NP(FAMR6G-ROX) with Ramos cells. In all cases, no significant fluorescence was displayed for the control cells. Next, we further tested whether each NP-conjugated aptamer could specifically identify target cancer cells from a mixture of three cells. The experiments were divided into two parts. In the first part of the experiment, before incubation with NP(FAM)-T1 conjugates, the control cells of CEM and Ramos were labeled with Cy5-sgc8 and Cy5-TDO5, respectively, then mixed with the

Figure 4. Confocal microscopy images of a mixture of three types of cells (target: Toledo; control: CEM and Ramos) incubated with one specific NP-aptamer conjugate NP(FAM)-T1. Control cells of CEM and Ramos were previously stained red with Cy5-labeled aptamer for differentiation from target Toledo cells. Only target Toledo cells displayed as blue from the label of NP(FAM)-T1.

Figure 5. Confocal microscopy images showing a mixture of three cells (Toledo, CEM, and Ramos) incubated with one type of NP-aptamer conjugate: (A) NP(FAM)-T1, specific for Toledo; (B) NP(FAM-R6G)-sgc8, specific for CEM; and (C) NP(FAM-R6GROX)-TDO5, specific for Ramos cells.

target Toledo cells in a ratio of 1:1:1. After incubating with the NP(FAM)-T1 for 20 min, the sample was washed twice by centrifugation to remove free aptamer-conjugated NPs that did not bind to the cells. From the fluorescence confocal image, two different cell populations can be clearly observed, one displaying blue (Toledo) for ∼1/3 and the other displaying red (CEM and Ramos) for ∼2/3, verifying that the NP(FAM)-T1 conjugates specifically associated with Toledo cell surfaces (Figure 4). In the second part of the experiment, we further added each NP-aptamer conjugate into a mixture with three cell types (CEM, Ramos, and Toledo, mixed in a ratio of 1:1:1). As shown in Figure 5, it can be clearly seen that each NP-aptamer conjugate could specifically bind to its corresponding target cell (NP(FAM)-T1 with Toledo (Figure 5A), NP(FAM-R6G)-sgc8 with CEM (Figure 5B), and NP(FAM-R6G-ROX)-TDO5 with Ramos (Figure 5C)). Similarly, the flow cytometry results also indicated that, as the percentage of target cells decreases in the mixture (from 100% pure target cells to 100% control cells), two clear populations could be observed with higher fluorescence for the target cells and lower fluorescence for the remainder. Also, with the increase of control cells (from 0% to 100%), the peak intensity of the NP-cell complex

Figure 6. Flow cytometry analysis of a three-cell mixture (target and control) using NP-aptamer conjugates: (A) Toledo cells (target), CEM and Ramos (control), and NP(FAM)-T1 conjugate; (B) CEM cells (target), Toledo and Ramos (control), and NP(FAM-R6G)-sgc8 conjugate; and (C) Ramos (target), Toledo and CEM (control) and NP(FAM-R6G-ROX)-TDO5 conjugate. Black line, 100% target cells; red line, 100% target; green line, 80% target + 20% control; dark blue line, 60% target + 40% control; pink line, 50% target + 50% control; light blue: 40% target + 60% control; yellow line: 20% target + 80% control; maroon line: 100% control. Black line corresponds to cells without any NP conjugates. The region from the red line to the maroon line represents cells incubated with NP-aptamer conjugates.

(at higher fluorescence) gradually decreases as a consequence of a lower amount of target cells, and the peak population in the lower fluorescence becomes gradually higher (Figure 6). This behavior could be observed with the three different NPs monitored in the three different emission channels in the flow cytometry. Moreover, an additional experiment in which three NP-aptamer mixtures (1:1:1) were put into single-cell solution (CEM, Ramos, or Toledo, respectively), it was observed that only the color of the cell-specific aptamer-labeled NP was observed in the confocal image (data not shown). Taken together, these results confirmed the powerful capabilities of these NPs-aptamer conjugates, both in terms of fluorescent signal and high selectivity toward target cells, thus showing the potential of their use for multiplexed, cancer cell analysis. Simultaneous Monitoring of Multiple Cancer Cells. To apply these FRET NP-aptamer conjugates to simultaneous monitoring of multiple cancer cells, different experiments were performed under confocal microscopy. First, a simplified experiment was performed using only a mixture of two different cells with the two corresponding NP-aptamer conjugates. The two cell types were mixed, and the corresponding NP-aptamer conjugates were simultaneously added. After incubating for 20 min, confocal images were taken upon excitation with the single wavelength of 488 nm. As expected, two distinct cell lines were observed with differentiated colors (see Figure S1 in the Supporting Information). For example, in the mixtures of Toledo and CEM cells, with NP(FAM)-T1 and NP(FAM-R6G)-sgc8, Toledo displayed the blue color of NP(FAM) and CEM displayed a light green color, coming from the NP(FAM-R6G), respectively (see panel A in Figure S1 of the Supporting Information). Subsequently, mixtures of three cell types, at a ratio of 1:1:1, were incubated with the three different NP-aptamer conjugates, following an analogous procedure with the same visible results (see Figure 7). That is, the three cancer cells were specifically covered with their corresponding aptamer-labeled NPs, and they show the distinct colors of the attached NPs. To demonstrate the applicability of the multiplexed analysis technique based on the FRET NP-aptamer conjugates in a more Analytical Chemistry, Vol. 81, No. 16, August 15, 2009

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Figure 7. Confocal images of a three-cell mixture incubated with the three NP-aptamer conjugates in binding buffer.

single-wavelength laser. When compared with single dyes, this fact, together with the higher signal amplification provided by the fluorescent NPs, clearly offers attractive advantages when sensitive simultaneous detection of multiple target cells is needed. Furthermore, with the capability of producing specific aptamers for virtually any type of cells, we believe this methodology can be developed for the multiplexed detection of any type of cancer cells. Therefore, work is currently in progress to extend this technique to other tumor cells, such as lung and ovarian cancer cell lines.

realistic biological environment, cell mixtures were suspended in cell medium (RPMI 1640) that contained 10% fetal bovine serum (FBS) and was incubated with NP-aptamer conjugates under similar conditions. Even in this case, an efficient binding of the corresponding NP-aptamer conjugates to the target cells could be observed by confocal microscopy (see Figure S2 in the Supporting Information).

ACKNOWLEDGMENT This work was supported by NIH, NCI, and NIGMS grants and by the State of Florida Center for Nano-Biosensors. X.L.C. acknowledges financial support from the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry. M.-C.E. acknowledges financial support from the Department d’Universitats, Recerca i Societat de la Informacio’ de la Generalitat de Catalunya, Spain.

CONCLUSION In summary, the results shown here demonstrate the feasibility of using fluorescence resonance energy transfer (FRET) silica nanoparticles (NPs) conjugated with aptamers for multiplexed monitoring of cancer cells. The system shows high specificity, which is provided by the properties of the aptamers immobilized on the surface of the NPs. Moreover, by changing the doping ratio of the dyes trapped inside the NPs, NPs with a variety of fluorescent emission spectra can be easily prepared and, more importantly, can be detected under the same excitation wavelength, simplifying the requirements of excitation source to a

SUPPORTING INFORMATION AVAILABLE Figures showing confocal images of two cells and two NP-aptamer mixtures (Figure S1) and a three-cell mixture incubated with the three NP-aptamer conjugates in cell media (Figure S2). (PDF) This material is available free of charge via the Internet at http://pubs.acs.org.

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Analytical Chemistry, Vol. 81, No. 16, August 15, 2009

Received for review May 21, 2009. Accepted June 20, 2009. AC9011073