Quantum Dots for Tumor-Targeted Fluorescence Imaging - American

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One-Pot Synthesized Aptamer-Functionalized CdTe:Zn2+ Quantum Dots for Tumor-Targeted Fluorescence Imaging in Vitro and in Vivo Cuiling Zhang,† Xinghu Ji,† Yuan Zhang,‡ Guohua Zhou,† Xianliang Ke,‡ Hanzhong Wang,‡ Philip Tinnefeld,§ and Zhike He*,† †

Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, P. R. China ‡ State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, P. R. China § Physical and Theoretical Chemistry, NanoBioScience, TU Braunschweig, Hans-Sommer-Strasse 10, 38106 Braunschweig, Germany S Supporting Information *

ABSTRACT: High quality and facile DNA functionalized quantum dots (QDs) as efficient fluorescence nanomaterials are of great significance for bioimaging both in vitro and in vivo applications. Herein, we offer a strategy to synthesize DNA-functionalized Zn2+ doped CdTe QDs (DNA-QDs) through a facile one-pot hydrothermal route. DNA is directly attached to the surface of QDs. The as-prepared QDs exhibit small size (3.85 ± 0.53 nm), high quantum yield (up to 80.5%), and excellent photostability. In addition, the toxicity of QDs has dropped considerably because of the Zn-doping and the existence of DNA. Furthermore, DNA has been designed as an aptamer specific for mucin 1 overexpressed in many cancer cells including lung adenocarcinoma. The aptamer-functionalized Zn2+ doped CdTe QDs (aptamer-QDs) have been successfully applied in active tumor-targeted imaging in vitro and in vivo. A universal design of DNA for synthesis of Zn2+ doped CdTe QDs could be extended to other target sequences. Owing to the abilities of specific recognition and the simple synthesis route, the applications of QDs will potentially be extended to biosensing and bioimaging.

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nanomaterials have been developed.12−20 Besides, metal-doped QDs, for instance, Mn2+ doped CdSe QDs and Zn2+ doped CdTe QDs by aqueous-phase synthesis have showed low cellular toxicity, high quantum yield, excellent biocompatibility, and small size.21,22 However, these QDs are sensitive to the experimental conditions, and it is difficult to functionalize them with DNA. More recently, DNA has been demonstrated to be unique ligands for the aqueous synthesis of QDs.23−26 Especially, cadmium-based QDs is one of the most studied systems for DNA-templated synthesis. The small, stable, and water-soluble cadmium-based QDs can be generated in one step, and the DNA molecules on the QD surface possess biorecognition capabilities that can be directly applied to specific targeting.25,27 The functionalized QDs have been widely applied in biosensing, bioimaging, and self-assembly.28−31 However, the QDs containing the highly toxic heavy metal element (cadmium)32 have low quantum yield and low output (400 μL of product at 4 μM), which are still the limitation for biomedical applications. Thus, an improved method to prepare DNA labeled QDs which meets the request of perfect QDs with small size, low

luorescence imaging is commonly used as one of the most potent tools for tumor-targeted imaging from cells and tissues of living animals.1−3 Functionalized quantum dots (QDs), which are modified and doped with biomolecules, have been widely applied in fluorescence imaging, and it is necessary and important that these have good biocompatible, specific targeting, and excellent fluorescence properties. DNA functionalized QDs are among the most studied functionalized QDs for fluorescence imaging. A common approach for obtaining DNA functionalized QDs is via simple avidin−biotin interaction due to its selectivity and strong binding (Kd = 10−15 M).4 However, it is difficult to control the binding site of a given biotin unit and the orientation of the biotin-appended DNA on the QD, because avidin has four binding sites. Also, the cross-linking and large size of avidincoated QDs are unfavorable for fluorescence imaging applications.5−8 Moreover, the previous investigations of our group have revealed that the large-size QDs could hardly enter cells.9,10 It has been realized that the optimal QDs for fluorescence imaging exhibit continuous light emission and have a size of 4−15 nm as well as low cytotoxicity.11 Core/shell structure and environmentally friendly materials are considered to be the potentialities due to the low toxicity. To date, many new types of QDs such as core/shell (CdSe/ZnS, CdTe/ZnS), core/shell/shell (CdTe/CdS/ZnS, CdSe/CdTe/ ZnSe), and environmentally friendly CuInSe, Ag2S, and Si QDs © XXXX American Chemical Society

Received: February 26, 2013 Accepted: May 20, 2013

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toxicity, and good fluorescence properties is ultimately needed for fluorescence imaging. Herein, labeling DNA on Zn2+ doped CdTe QDs (DNAQDs) by the one-pot method has been accomplished. The QDs have high quantum yield (up to 80.5%) and high output (2 mL of product at 30 μM). Because of the doping with Zn, the QDs have reduced toxicity. The unique functional domain of DNA on CdTe:Zn2+ QDs is designed as the aptamer which can recognize mucin 1. Mucin 1 has been widely overexpressed in human lung adenocarcinoma cells.33 In this research, the A549 cells (human lung adenocarcinoma epithelial cell line) were chosen as the target cell. It is worth mentioning that the aptamer-QDs have been designed for the first time for application in active tumortargeted imaging in vitro and in vivo.

estimated by the absorption spectra according to a reported method.35 The concentration of DNA was calculated from the absorbance at 260 nm after subtracting the contribution of QDs, for QDs make a contribution to the absorbance at 260 nm. The molar extinction coefficient of DNA is 2 × 105 M−1 cm−1 by the synthesis report of DNA. Then, the DNA to QDs ratio was calculated accordingly. Hybridization between DNA-QDs and Complementary DNA. The hybridization reaction was performed by mixing DNA-QDs and the complementary DNA. First, 0, 5, and 10 μL of 1 μM complementary DNA in 10 mM PBS buffer solution (pH 7.4, 15 mM NaCl) were added into DNA-QDs solution. Then, the buffer solution was added into a final volume of 500 μL and held about 30 min. The final concentration of QDs was 8 nM. Twenty μL of SYBR green 1 (diluted 10 000 times of the original solution) was added and held about 5 min. At last, the fluorescence signals of QDs and SYBR green 1 were detected. Cell Cytotoxicity Assay. The cytotoxicity of DNA-QDs was evaluated by the MTT assay. A549 cells were cultured in 96 well plates at a density of 2500 cells per well in 200 μL of DMEM and then cultured 16 h at 37 °C. QDs (CdTe, CdTe:Zn2+, CdTe:Zn2+-DNA) were added to each well to a final concentration of 0, 0.375, 0.75, 0.15, 0.3, 0.6, 1.2, and 2.4 μM. 96 well plates were incubated for 24 h. After that, the supernatant was replaced with fresh DMEM (200 μL). Twenty μL of 5 mg L−1 MTT was added to each well, and then, the PBS buffer solution was added to a final volume of 200 μL. The mixture was removed after being incubated for 4 h; 200 μL of DMSO was added to each well to dissolve formazan crystals by rude shock for 1 min. The absorbance at 570 nm was measured using a microplate reader (Bio-Rad550, USA). Data was collected in quadruplicate, and the final data was averaged. The percent relative viability of A549 cells related to the control well containing PBS buffer without QDs was calculated by the following equation:

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EXPERIMENTAL SECTION Chemicals and Materials. NAC (98%), rhodamine 6G, sodium phosphate monobasic dihydrate (NaH2PO4·2H2O), sodium phosphate dibasic dodecahydrate (Na2HPO4·12H2O), sodium chloride (NaCl), dimethyl sulfoxide (DMSO), and Hoechst 33342 were commercially available from Sigma (USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was bought from Amresco (USA). Dulbecco’s modified Eagle medium (DMEM) was purchased from Gibco Inc. CdCl2·2.5H2O, ZnCl2, tellurium powder (99.999%), and sodium borohydride (NaBH4) were obtained from Sinopharm Chemical Reagent (China) and were used as received without additional purification. All chemicals used were of analytical grade or of the highest purity available. Modified DNA, 5′-TCCGCTGCAGAAAAA AAT*C*G*G*G*C*G*T*A*C-3′ (* indicates the phosphorothioate linkage), modified aptamer, GCAGTTGATCCTTTG GATACCCTGGAAAA AAT*C*G*G*G*C*G*T*A*C-3′, complementary modified DNA, 5′-TTCTGCAGCGGA-3′, and SYBR green 1 were synthesized by Shanghai Sangon Biotechnology Co. Ltd. (Shanghai, China). The mucin 1 with two repeats of the 20 amino acid variable tandem repeat region (from the N terminus to the C terminus: PDTRPAPG STAPPAHGVTSAPDTRPAPGSTAPPAHGVTSA) was synthesized by the ChinaPeptides Co., Ltd. (Shanghai, China). All solutions were prepared using ultrapure water obtained from the Milli-Q system (Millipore, USA). Preparation of DNA Functionalized Zn Doping CdTe Nanocrystals. The DNA-QDs solution was synthesized by reacting NaHTe with Cd2+ and Zn2+, in which NAC and modified DNA are the stabilizing reagents at pH 9.0. The fresh NaHTe solution was obtained by the reaction of 25 mg of Te power and 20 mg of NaBH4 in 1 mL of water at 0 °C for 5 h. Then, the fresh NaHTe solution was injected into 2 mL of oxygen-free aqueous solution containing CdCl2·2.5H2O and ZnCl2 at pH 9.0. Sequentially, DNA solution containing 1.5 μM nucleotides was added to the mixture. The molar ratio of Cd, Zn, Te, and NAC used was 1:2:0.2:3.6. Last, the mixture solution was transferred to a Teflon lined stainless steel autoclave and heated to the growth temperature (200 °C). In order to get the pure QDs at the end of the synthesis, the reaction solution was purified by ultrafiltration using an Amicon Ultra-4 centrifugal filter device with a MW cut off of 30 kDa (Millipore Corp., USA) The as-prepared products were stored at 4 °C or lyophilized to obtain the QD powder for characterization. The quantum yields of QDs were determined by rhodamine 6G (95%, as reference standard) and rhodamine B (98%, as reference standard).34 The labeling efficiency was calculated as follows: the concentrations of CdTe:Zn2+ QDs and DNA-QDs were

cell viability (%) = (A sample−A blank )/(Acontrol − A blank ) × 100

(1)

where Asample is the absorbance of the solution containing cells cultured with QDs, Ablank is the absorbance of the PBS buffer, and Acontrol is the absorbance of the cells only. DNA-QDs Applied in Tumor Targeting in Vitro and in Vivo. First, the nuclei of A549 cells and Vero cells were stained with Hoechst 33342 simply by adding the dye (5 μg/mL, 30 min) to the DMEM with 10% heat-inactivated fetal bovine serum (FBS) culture medium, and the cells were washed three times. Second, the cells were cultured (37 °C, 5% CO2) with 200 nM aptamer-QDs (4 °C, 30 min, and 45 min) or first 600 nM aptamer (4 °C, 30 min) and then 200 nM aptamer-QDs (4 °C, 30 min). Then, the cells were washed three times with buffer. Finally, the fluorescence images were measured on the UltraView VOX confocal system (PerkinElmer, USA) attached to an inverted microscope (Nikon, Japan) with 488 nm laser excitation; 100× objective lenses were chosen for observation. The images of QDs interaction with cells were dealt with the Volocity software. The tumor-bearing nude mice were supplied by Wuhan BioMed Science & Technology Co. Ltd. (China) The sizes of tumors were about 0.5 cm3. Two nmol of aptamer QDs or nonaptamer QDs was injected into a nude mouse by tail vein injection. Then, mice were anesthetized with ketamine (40 mg/ B

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spherical particles and well dispersible (Figure 2A). The highly crystalline structure of the DNA-QDs was visualized on the high resolution TEM (HRTEM) image (inset in Figure 2A). As shown in Figure 2B, X-ray diffraction (XRD) characterization exhibited that the prepared DNA-QDs belonged to the cubic zinc blende structure. A size distribution histogram of DNA-QDs (λem: 574 nm) was obtained by measuring more than 300 particles, which revealed the particle sizes of DNA-QDs of 3.85 ± 0.53 nm (Figure 2C). Dynamic light scattering (DLS, Figure 2D) was performed to determine the hydrodynamic diameter of the QDs. In comparison, the corresponding average hydrodynamic diameter of the DNA-QDs in water was about 7.3 nm. The difference in diameter measured by TEM and DLS should be attributed to the surface DNA ligand of the DNA-QDs in aqueous solution. Most importantly, the small size of as-prepared DNA-QDs is advantageous for bioimaging compared to the DNA-labeled QDs bridged by avidin−biotin. Availability of DNA on the CdTe:Zn2+ QDs. The availability of DNA on the QDs was tested by hybridization with complementary DNA in combination with SYBR green 1 staining (Figure S1, Supporting Information). SYBR green 1 is a fluorescent asymmetrical cyanine dye whose fluorescence is very weak in aqueous solution. The fluorescence is dramatically increased when it binds to double stranded DNA (dsDNA) but not single stranded DNA (ssDNA).37 By this strategy, the dsDNA was formed by hybridizing the DNA on the surface of QDs with the complementary DNA, and when SYBR green 1 was added, the fluorescence of it was increased sharply. The more complementary DNA was added, the stronger was the fluorescence intensity of SYBR green 1 observed. All of these results have verified the availability of the DNA on the surface of QDs. UV−Vis Absorption and Fluorescence Spectra of DNAQDs and CdTe:Zn2+ QDs. DNA-QDs and CdTe:Zn2+ QDs show similar UV−vis absorption and fluorescence spectra, indicating that the DNA does not negatively influence the luminescence properties of the QDs (Figure S2, Supporting Information). The absorption at 260 nm of DNA-QDs is attributed to DNA, further supporting that the DNA is attached to the QDs. In addition, the spectrum of the DNA-QDs is blueshifted by 3 nm compared with CdTe:Zn2+ QDs. This result indicates that the DNA ligand may interact electronically with the crystal surface and alter its electronic properties.28,29 Optical Properties of the As-Prepared DNA-QDs. DNAQDs with different colors are prepared by controlling the reaction time. Figure 3A,B shows the UV−vis spectra and the fluorescence spectra of a series of as-prepared DNA-QDs with maximum emission wavelengths ranging from 546 to 646 nm, respectively. The corresponding emission decay times of DNAQDs display a gradual increase from 23.7 ± 1.0 to 34.0 ± 1.2 ns as the emission wavelength increased (Figure 3C). Overall, the asprepared DNA-QDs exhibit a high quantum yield (up to 80.5%), long fluorescence decay times, and narrow emission bands (see photographs in Figure 3D and Table S1 in Supporting Information). Furthermore, we could obtain 2 mL of DNAQDs solution at a concentration of 30 μM by the one-pot method. This up scaling is important to meet the need of quantity in biological applications. Figure 4 shows the photostability result. The DNA-QDs and CdTe QDs solutions were irradiated continuously over a period of 60 min. The fluorescence intensity of CdTe QDs solution only remained at 35% of the initial value, while the fluorescence intensity of DNA-QDs stabilized at around 62%. This suggested

kg). After 10 min, mice were imaged with the Maestro in vivo imaging system (Cambridge Research & Instrumentation, USA). Apparatus and Characterization. UV−vis absorption spectra were obtained by a Shimadzu UV-2550 spectrophotometer (Japan). Fluorescence spectra were measured on a Shimadzu RF-5301 fluorescence spectrophotometer (Japan). The FT-IR spectra were recorded on a Perkin-Elmer-2 spectrometer (USA). A TEM sample was made by dropping QD aqueous solution onto ultrathin carbon-coated copper grids. TEM images were conducted on a JEM 2100 transmission electron microscope (Japan). A dynamic light scattering (DLS) measurement was measured on the Zetasizer instrument ZEN3600 (Malvern, UK) with a 173° back scattering angle and He−Ne laser (λ = 633 nm). The crystal phase of QDs was characterized by a Bruker D8 Discover X-ray Diffractometer (German) with 2θ range from 10° to 70° at a scanning rate of 2° per minute. The interactions between aptamer and mucin 1 protein were characterized on the chirascan dichroism spectrometer (Applied Photophysics, UK).

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RESULTS AND DISCUSSION The design strategy is illustrated in Figure 1. The one-pot hydrothermal process permits the preparation of high zinc

Figure 1. Schematic illustration of structures of (A) phosphorothioates and functional domain and (B) the one-pot synthesized DNA-QDs route.

content, good photostability, and high quantum yield DNA-QDs with antioxidant N-acetylcysteine (NAC) ligand. DNA was used as the coligand to obtain the DNA functionalized QDs (Figure 1B). This DNA ligand contains three domains, phosphorothioates which bind to QDs because of their high affinity to cadmium, linker, and functional domain. Structures of phosphorothioates and functional domain are shown in Figure 1A. In the present synthesis strategy, DNA is crucial to the growth of QDs. Amine and carbonyl groups are responsible for surface passivation; steric and/or secondary conformation effects of the capping strands play an important role in stabilizing the colloids, and overall surface reactivity of the sequences mainly influences the quality of nanocrystals.25,26,36 During the synthesis process, the CdTe:Zn2+ QDs were gradually formed, and the S atoms in the phosphorothioates domain of the designed DNA were inserted into CdTe:Zn2+ QDs. However, functional domain (nonphosphorothioates) extended away from the surfaces of CdTe:Zn2+ QDs and was available for recognition to the complementary DNA, peptide, or protein. Crystal Structure and Hydrodynamic Diameter Characterizations of DNA-QDs. The transmission electron microscopy (TEM) image had shown that the DNA-QDs were C

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Figure 2. (A) TEM image (inset: the HRTEM image); (B) the XRD of the prepared DNA-QDs (λ em = 574 nm), the standard pattern of CdTe QDs (JCPDs card 15-0770) is also offered at the bottom lane; (C) the size distribution; (D) dynamic light scatting histogram.

Figure 3. (A) UV−vis spectra, (B) fluorescence spectra, and (C) fluorescence lifetime decay curves of DNA-QDs with controllable maximum emission wavelengths ranging from 546 to 646 nm. (D) Photographs of the aqueous solution of DNA-QDs under UV irradiation and visible light conditions by a digital camera. The samples were directly extracted from the original solution right after reaction without further treatment.

that the DNA-QDs had a better photostability for tracing over extended time periods as well as for imaging. Controllability Numbers of DNA Molecules per QD. Under the same conditions, the numbers of DNA per QD are

different with different amounts of DNA added (Figure S3, Supporting Information). In order to vary the number of DNA molecules per QD, different amounts of initial DNA were added. The numbers of DNA sequences per QD were determined to be D

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same concentration was only 7%. That is, DNA-QDs have the lowest toxicity within this series. Besides, the toxicity of CdTe QDs has been discussed comprehensively in previous research;32 0.3 μM of CdTe QDs could lead to 80% of cell death, while 0.3 μM of DNA-QDs could keep 50% of cells viable. There are two reasons for this phenomenon. First, high Zn doped ratio (the surface molar ratio of Zn to Cd is 1:1) plays a key role. Second, DNA capping may also reduce toxicity, for that can improve the stability and prevent the release of toxic components.40,41 All of these results indicated that DNA-QDs showed a promising prospect in bioimaging application. Characteristics of Interactions between Aptamer and Mucin 1 by Circular Dichroism. Mucin 1 is overexpressed over 90% in late stage epithelial ovarian cancers and metastatic lesions but not in normal ovarian tissues.42 The DNA aptamer specific for mucin 1 was previously reported to assist doxorubicin enter cells for treatment of multidrug resistant ovarian cancer. It is therefore an interesting candidate that was used as the recognition domain of DNA. For the following experiments, we synthesized aptamer-QDs (the recognition area of DNA was designed as the aptamer of mucin 1) which could have specific binding with mucin 1, which was known to be expressed on the cell surface of A549 cells.43 To clarify the interactions between aptamer and mucin 1, the circular dichroism (CD) experiment was performed. Figure S5 (Supporting Information) has shown the CD spectra of aptamer-QDs, mucin 1, aptamer-QDs-mucin 1 complex, and aptamer-mucin 1 complex. Aptamer-QDs have almost no absorption at the far ultraviolet region. The CD spectrum of the 40-amino acid synthetic peptide (2 repeats) contains a large negative peak at around 206 nm. This spectrum is characteristic of proline-rich repeat proteins.44,45 When aptamer or aptamer-QDs were added to the mucin 1 solution, the negative peak decreased, which demonstrated that the structure of mucin 1 was destroyed because of interactions between aptamer and mucin 1. In Vitro and in Vivo Targeted Imaging. To demonstrate the aptamer-QDs as tumor cellular target probes, we further employed our aptamer-QDs for in vitro imaging. The aptamerQDs were first evaluated for tumor targeting in this paper. The confocal images confirmed strong binding of the aptamer-QDs to A549 cells (Figure 6A). The aptamer-QDs were mainly distributed near the membrane within a 30 min incubation time at 4 °C. When the incubation time lasted longer (45 min), these had been spread mostly to cytoplasm and near nuclear membrane (Figure S6, Supporting Information). A control experiment using Vero cells, mucin 1-negative kidney cells, showed little binding to the membrane (Figure 6B). Additional control experiment using DNA-QDs, which were not linked with mucin 1 aptamer but with a random sequence, also showed the absence of binding (Figure 6C). The specific interaction experiment was done by pretreating A549 cancer cells with aptamer and then incubating with the aptamer-QDs (Figure 6D); there was almost no fluorescence. From such results, aptamer-QDs could be applied in tumor targeting in vitro. To the best of our knowledge, this is the first report of evaluating aptamer-QDs which were synthesized by a one-pot hydrothermal method for tumor-targeted imaging. To further investigate the feasibility of aptamer-QDs in vivo, we conducted lung cancer tumor targeting and imaged in live animals. To start with, the aptamer-QDs and nonaptamer QDs were intravenously injected into tumor-bearing nude mice. Figure 7 shows fluorescence images of the tumor-bearing mice injected with different QDs. No signals were observed from the

Figure 4. Photostability of DNA-QDs (black) and CdTe QDs (red) (Xe lamp, 150 W, Ex: 350 nm).

0.36, 1.3, 1.83, and 2.67 (when the added ratios of DNA are 1:1,1.3:1, 1.7:1, and 2.6:1, respectively) using UV absorption of DNA-QDs and CdTe:Zn2+ QDs. It suggested that the numbers of DNA per QD could be one or two by controlling the amount of DNA. In Vitro Cytotoxicity. Cytotoxicity assessment of QDs is another critical consideration for the bioimaging application. In the present study, we used the 3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide (MTT) assay to estimate the cytotoxicity at various concentrations of CdTe QDs, CdTe:Zn2+ QDs, and DNA-QDs. This method has been widely used to quantify cell death of A549 cells.38,39As shown in Figure 5, the CdTe QDs were obviously the most toxic, and the DNA-QDs were less toxic than CdTe:Zn2+ QDs. Fortunately, the A549 cells maintained more than 40% viability following the treatment with DNA-QDs at concentrations as high as 2.4 μM. In contrast, the viability of A549 cells treated with CdTe:Zn2+ QDs degraded to 14%, and the viability of A549 cells treated with CdTe QDs at the

Figure 5. Cytotoxicity of A549 cells incubated with different concentrations of DNA-QDs (a), CdTe:Zn2+ QDs (b), and CdTe QDs (c) for 24 h. E

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CONCLUSIONS In this study, DNA functionalized Zn-doped CdTe QDs were directly synthesized through a facile one-pot hydrothermal route. The QDs exhibit high quantum yield, low cellular toxicity, small size, excellent photostability, and biocompatibility. Most importantly, the as-prepared aptamer-QDs were well suitable for in vitro and in vivo imaging of tumor targeting. Compared to other methods of DNA functionalized QDs, these DNA-QDs are more adaptable for the further study of highly specific and sensitive bioimaging. Such DNA-QDs are promising tools for disease diagnosis.

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ASSOCIATED CONTENT

S Supporting Information *

Details for fluorescence spectra, UV−vis spectra, and FT-IR characterizations. 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].

Figure 6. Confocal fluorescent tumor cellular target studies of QDs. (A) A549 cells, which are mucin 1-positive, as shown in the presence of the CdTe:Zn2+-aptamer QDs (aptamer-QDs) binding to the cell surface; (B) the negative control was noted in Vero cells that lack mucin 1 expression incubated with aptamer-QDs; (C) the negative control was detected in A549 cells incubated with DNA-QDs which were not linked with mucin 1 aptamer but with a random sequence; (D) A549 cells were first incubated with aptamer and then aptamer-QDs. Scale bars: 7 μm.

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Key Scientific Program Nanoscience and Nanotechnology (2011CB933600), the National Science Foundation of China 425 (21275109, 21075093), Large-scale Instrument and Equipment Sharing Foundation of Wuhan University,and academic award for excellent Ph.D. Candidates funded by Ministry of Education of China 427 (5052012203001).

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REFERENCES

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Figure 7. (A) Fluorescence images and (B) photographs of tumorbearing mice injected with aptamer-QDs (2 nmol, left) and nonaptamer QDs (right). Tumor organs of the mice were also analyzed (insets in B). The autofluorescence of the mouse was removed by spectral unmixing. All images were obtained under the same experimental conditions.

tumor-bearing mouse treated with nonaptamer QDs; on the contrary, strong signals located at the tumor could be seen from the mouse treated with aptamer-QDs. This finding is substantiated by fluorescence images of tumor organs that also showed higher accumulation of the targeting aptamer-QDs (Inset in Figure 7B). All of these data demonstrated that the aptamer-QDs had high selectivity and sensitivity for active tumor targeting in vivo. These results reveal that the aptamer-QDs can serve as efficient probes for future tumor diagnosis applications. F

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dx.doi.org/10.1021/ac400606e | Anal. Chem. XXXX, XXX, XXX−XXX