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Breast cancer cells synchronous labeling and separation based on aptamer and fluorescence-magnetic silica nanoparticles
Optical Materials 75 (2018) 483e490
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Optical Materials journal homepage: www.elsevier.com/locate/optmat
Breast cancer cells synchronous labeling and separation based on aptamer and fluorescence-magnetic silica nanoparticles Qiu-Yue Wang a, Wei Huang b, Xing-Lin Jiang a, Yan-Jun Kang b, * a b
Hunan University of Medicine, Huaihua, Hunan 418000, PR China Wuxi School of Medicine, Jiangnan University, Wuxi, Jiangsu 214122, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 July 2017 Received in revised form 28 October 2017 Accepted 3 November 2017
In this work, an efficient method based on biotin-labeled aptamer and streptavidin-conjugated fluorescence-magnetic silica nanoprobes (FITC@Fe3O4@SiNPs-SA) has been established for human breast carcinoma MCF-7 cells synchronous labeling and separation. Carboxyl-modified fluorescence-magnetic € ber method. Strepsilica nanoparticles (FITC@Fe3O4@SiNPs-COOH) were first synthesized using the Sto tavidin (SA) was then conjugated to the surface of FITC@Fe3O4@SiNPs-COOH. The MCF-7 cell suspension was incubated with biotin-labeled MUC-1 aptamer. After centrifugation and washing, the cells were then treated with FITC@Fe3O4@SiNPs-SA. Afterwards, the mixtures were separated by a magnet. The cellprobe conjugates were then imaged using fluorescent microscopy. The results show that the MUC-1 aptamer could recognize and bind to the targeted cells with high affinity and specificity, indicating the prepared FITC@Fe3O4@SiNPs-SA with great photostability and superparamagnetism could be applied effectively in labeling and separation for MCF-7 cell in suspension synchronously. In addition, the feasibility of MCF-7 cells detection in peripheral blood was assessed. The results indicate that the method above is also applicable for cancer cells synchronous labeling and separation in complex biological system. © 2017 Elsevier B.V. All rights reserved.
Keywords: Fluorescence Magnetism Silica nanoprobes Aptamer Streptavidin MCF-7 cells
1. Introduction Breast cancer is one of the commonest malignant tumors in women. According to the statistics, there are about 1.2 million new patients with breast cancer each year in the world, of which 0.5 million patients died of breast cancer. Tumor metastasis is the leading cause of death in patients. Circulating tumor cell (CTC) is a kind of tumor cell that exists in peripheral blood, which spreads to other organs of the body by the blood circulation [1]. However, most of CTCs in peripheral blood suffer from apoptosis or cytophagy. Very few of CTCs escape and develop into metastatic lesion, and increase the mortality risk of patients. Therefore, the early detection of breast cancer CTC in the blood would play important guiding roles in the terms of monitoring of tumor recurrence, therapeutic evaluation, and individual treatment. At present, CTCs are mainly detected using immunocytochemistry (ICC), Flow cytometry (FCM), and RT-PCR [2e4]. Briefly, ICC reflects the existence and distribution of biomacromolecules such
* Corresponding author. E-mail address: [email protected] (Y.-J. Kang). https://doi.org/10.1016/j.optmat.2017.11.003 0925-3467/© 2017 Elsevier B.V. All rights reserved.
as proteins, membrane surface antigens, and receptors, but the procedure is tedious and time-consuming. In addition, the poor photostability of fluorescein may influence the observation results. Both FCM and RT-PCR are sensitive and fast, which can quantitatively analyze the cell components such as DNA. However, cell morphology and more biological information can't be acquired by the above methods. How to develop an efficient and sensitive method for CTCs detection in complex biological system is essential in bioanalysis. With the development of nanotechnology in recent years, some fluorescent nanomaterials such as quantum dots, carbon dots, and dye-doped silica nanoparticles have showed great advantages in the field of biomedical imaging because of their unique optical properties [5e7]. In addition, the AgInZnS nanoparticles used for cell labeling is also reported [8]. However, faced with the complex system, these materials only play a role of single labeling. Other substances in the mixtures may reduce the labeling efficiency of targets and disturb the signals of detection. To enhance the sensitivity, the process of pretreatment and purification is necessary for most fluorescent-labeling based methods. In addition, although magnetic nanomaterials such as a-Fe, Fe3O4, and aFe2O3 have aroused great concern in the field of separation science
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[9e11], they mainly provide single separation. Besides, some magnetic materials are prone to aggregation after the removal of external magnetic field due to the hysteresis, which results in poor separation of targets. Moreover, the complex procedure of preparing biomolecules-functionalized magnetic nanoprobes and poor biocompatibility of bare magnetic nanomaterials should be considered [12]. Therefore, it is urgent to prepare composite nanomaterials with fluorescence, superparamagnetism, and good biocompatibility for targets synchronous labeling and separation. Composite silica nanomaterials have attracted considerable attention in biomedical analysis because of their antiphotobleaching, larger specific surface area, small size, good biocompatibility, facile surface modification, and special magnetic properties [13e15]. Especially, dye-doped silica nanoparticles have been widely used for cancer cells labeling, DNA microanalysis, and the detection of pathogenic bacteria [16e18]. In addition, Fe3O4enwrapped silica nanoparticles (Fe3O4@SiO2) with superparamagnetism could be aggregated immediately in the presence of external magnetic field and dispersed rapidly in solution after the removal of external magnetic field due to no hysteresis, which have been widely used for separation, and magnetic resonance imaging (MRI) [19e21]. In this paper, to prepare composite silica nanoprobes with fluorescence and magnetism for targets synchronous labeling and separation, Fe3O4 nanoparticles and fluorescent dyes (FITC) are enwrapped simultaneously into the silica € ber method [22,23]. Targeted nanoprobes networks using the Sto are then prepared by conjugating the recognizable biomolecules to the surface of fluorescence-magnetic silica nanoparticles. As we know, antibodies as the conventional recognition elements have been widely used for biolabeling. However, they can't meet the demands of modern biological analysis increasingly. Exploring promising recognition elements has always been a goal pursued by researchers. In recent years, the aptamer based on the systematic evolution of ligands by exponential enrichment (SELEX) has attracted great concern [24]. Aptamers are single-stranded nucleic with three-dimensional structure, which show high affinity and specificity with all kinds of targets such as cancer cells, pathogenic bacteria, Virus, ATP, and even metal ions [25e29]. Compared with antibodies, aptamers take advantages of strong tissue permeability, ignorable immunogenicity, high affinity and specificity with flexible targets, easy preparation and preservation, and small molecular weight. Inspired by the extraordinary properties of aptamer and fluorescence-magnetic silica nanoparticles, we develop an efficient and sensitive method based on biotinlabeled aptamer and streptavidin-conjugated fluorescence-magnetic silica nanoprobes for breast cancer cells in the blood synchronous labeling and separation. This technology not only sensitively, conveniently, real-timely, and dynamically indicates the tumor progression and molecular biology information but also plays important guiding roles in the terms of monitoring of tumor recurrence, therapeutic evaluation, and individual treatment. 2. Experimental 2.1. Chemicals and materials TritonX-100, tetraethyl orthosilicate (TEOS), (3-aminopropyl) triethoxysilane (APTES), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimid ehydrochloride (EDC), N-hydroxysulfosuccinimide sodium salt (sulfo-NHS), Fluorescein isothiocyanate (FITC), and Streptavidin were purchased from Sigma Chemical Co. (St. Louis. MO). Ammonium hydroxide (25-28 wt%), Ferric chloride hexahydrate (FeCl3$6H2O), Ferrous chloride tetrahydrate (FeCl2$4H2O), alcohol, N,-N-Dimethylformamide (DMF), and Succinic anhydride were purchased from China Pharmaceutical Group Shanghai
Chemical Reagent Co., Ltd. Human breast carcinoma MCF-7 cells lines were obtained from china center for type culture collection. FITC-labeled MUC-1 aptamer was synthesized from Beijing Biosynthesis Biotechnology Co.,Ltd. The biotin-labeled MUC-1 aptamer, and the biotin-labeled random ssDNA were synthesized in Shanghai Sangon Biological Engineering Technology & Services Co. (China), and the sequence as follows: 5/-biotin-(CH2)6-GGGAGACAAGAATAAACGCTCAAGCAGTTGATC CTTTGGATACCCTGGTTCGACAGGAGGCTCACAACAGGC-3/ 5/-biotin-(CH2)6-ACAGGAGGCTCACAAACGCTCAAACAGGAGGCT CACTAACGGACGTTCGACAGGAGGCTGGACGTTCGACGA-3/ 2.2. Main instrumentation The size and uniformity of synthesized nanoparticles were measured by means of transmission electron microscope (JEOL, JEM100CXII, Japan). Photoluminescence was measured using fluorescence spectrophotometer (F96PRO, China). Fluorescence images results were observed with under inverted fluorescence microscope (Nikon ECLIPSE TE2000-U, Japan). Magnetic characterization of synthesized nanoparticles was performed using vibrating sample magnetometer (Lakeshore 7410, USA). Structural analysis of prepared nanoprobe was performed using X-ray diffraction (XRD6000, SHIMADZU). 2.3. Fe3O4 nanoparticle synthesis Fe3O4 nanoparticles were prepared by the coprecipitation method. Briefly, 6.76 g of FeCl3$6H2O, 2.49 g of FeCl2$4H2O, and 200 mL of deionized water were added together to a flask. 0.4 M ammonia hydroxide was then gradually added to the iron chloride solution stirred at 350 rpm using a mechanical stirrer until pH reached 10.0. The reaction was allowed to continue for 30 min at 30 C, followed by centrifuging and washing with deionized water three times. The products were finally resuspended in 95% ethanol for the next step. 2.4. Preparation of FITC-APTES precursor 35 mL of APTES and 3 mg of FITC were mixed in 0.5 mL of absolute ethanol under dry nitrogen atmosphere and stirring magnetically for 12 h. In addition, the FITC-APTES conjugates solution was protected from light during reaction and storage. 2.5. Preparation of carboxyl-modified fluorescent-magnetic silica nanoparticles Carboxyl-modified fluorescence-magnetic silica nanoparticles € ber (FITC@Fe3O4@SiNPs-COOH) were synthesized using the Sto method. Briefly, 40 mL of ethanol, 10 mL of deionized water, and 0.5 mL of 50 mg/mL Fe3O4 were sonicated for 90 min and then stirred at 350 rpm using a mechanical stirrer. 1 mL of ammonia hydroxide and 200 mL of FITC-APTES were then added to the solution stirred. To create the silica coating, 1 mL of TEOS was added, and the mixture was stirred for 4 h to complete the hydrolysis process. For postcoating, 50 mL of TEOS and 50 mL of APTES were added and additional stir was performed for 2 h. By washing and resuspending with DMF solution, the products were reacted with succinic anhydride under nitrogen gas for 24 h with continuous stirring. Then the prepared FITC@Fe3O4@SiNPs-COOH were washed by water and resuspended in PBS for the next step. 2.6. Conjugation of streptavidin onto FITC@Fe3O4@SiNPs-COOH 0.5 mL of FITC@Fe3O4@SiNPs-COOH, 1 mg of EDC, and 2.5 mg of
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Sulfo-NHS were added to 2 mL of 0.1 M PBS buffer (pH 7.4). The mixture was then incubated for 15 min at room temperature with gentle shaking. After the reaction completed, 50 mL of 1 mg/mL streptavidin was immediately added to the solution, following by 3 h incubation with gentle shaking at room temperature. Particles were washed with 0.1 M PBS (pH 7.4) and then resuspended in 1 ml of 0.05% BSA for 1 h to block the free carboxylates. After the reaction completed, the prepared streptavidin-conjugated fluorescencemagnetic silica nanoparticles (FITC@Fe3O4@SiNPs-SA) were washed with 0.1 M PBS buffer (pH 7.4) three times and then resuspended in 0.1 M PBS (pH 7.4) for further use. 2.7. Cancer cells synchronous labeling and separation Human breast carcinoma MCF-7 cells were cultured until the cover rate on the six-well plate reached 70e90%. After the removal of the medium, the adherent cells were digested by pancreatin. Fresh medium was then added to the six-well plate, and the suspended cells were centrifuged (1000 rpm 5 min). After the removal of the supernatant, the collected cells were resuspended in 2 mL of 0.1 M PBS (pH 7.4). 100 mL of cell suspension was diluted 10fold with PBS. 100 mL of 10 mM biotin-labeled MUC-1 aptamer and cells suspension diluted were incubated for 60 min at 37 C with gentle shaking. After the reaction completed, the suspension of cells-aptamer complexes were centrifuged and washed with PBS three times, and then resuspended in 1 mL of 0.1 M PBS (pH 7.4). 50 mL of FITC@Fe3O4@SiNPs-SA was immediately added to the suspension. After incubated for 60 min at 37 C with gentle shaking, the mixtures were separated by a magnet and washed with 0.1 M PBS (pH 7.4) three times. The cell-nanoprobe conjugates were finally resuspended in PBS and then placed on the slide glass for fluorescence microscope observation. Corresponding control group was treated through the same procedure with exception of the biotin-labeled MUC-1 aptamer was replaced by biotin-labeled random ssDNA. 2.8. Cancer cells synchronous labeling and separation in peripheral blood 100 mL of MCF-7 cells in 0.1 M PBS (pH 7.4) with a concentration of 103 cell/mL was added to 1.5 mL of normal human peripheral blood sample in the presence of 2 mg of EDTA dipotassium salt. After mixed fully, 100 mL of 10 mM biotin-labeled MUC-1 aptamer was then added to the blood sample above. The samples were incubated for 60 min at 37 C with gentle shaking, and then centrifuged (4000 rpm 5 min). After the removal of the upper solution, 100 mL of FITC@Fe3O4@SiNPs-SA was immediately added to the residual suspension. After incubated for 60 min at 37 C with gentle shaking, the suspension was treated by a magnet and washed with 0.1 M PBS (pH 7.4) three times. The acquired products were resuspended in PBS, and finally placed on a slide for fluorescence microscopic imaging. The control groups follow the steps above except that the biotin-labeled random ssDNA was used instead of biotin-labeled MUC-1 aptamer. 3. Results and discussion 3.1. Strategy based on aptamer and fluorescence-magnetic silica nanoprobes for MCF-7 cells synchronous labeling and separation In this paper, we used the reported aptamer with 72 bases as the recognition element because of its high affinity and specificity to the MUC-1 protein on the surface of human breast carcinoma MCF7 cells [30,31]. To avoid the steric hindrance between cells and nanoprobes, the biotin-labeled aptamer and streptavidin-
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conjugated fluorescence-magnetic nanoprobe (FITC@Fe3O4@SiNPs-SA) were individually used instead of aptamer directly conjugated nanoprobes (FITC@Fe3O4@SiNPs-aptamer). The overall schematic is described in Fig. 1. The mixtures containing MCF7 cells were firstly treated with the free biotin-labeled MUC-1 aptamer. After incubated, the unbound biotin-labeled aptamer was removed by low-speed centrifugation and washing. Owing to the interaction between biotin and streptavidin, the targeted cells were subsequently captured by adding FITC@Fe3O4@SiNPs-SA to the resuspended cells. After incubated, the cell-probe complexes were separated by a magnet, and then placed on a slide for fluorescence microscopic imaging. The control groups follow the steps above except that the biotin-labeled random ssDNA was used instead of biotin-labeled MUC-1 aptamer. First of all, the aptamer as the promising recognition element was used instead of antibodies because of its high affinity and specificity with flexible targets, easy preparation and preservation, small molecular weight, strong tissue permeability, and ignorable immunogenicity. Moreover, compared with the methods of single separation and labeling, the fluorescence-magnetic silica nanoprobes (FITC@Fe3O4@SiNPs-SA) with superparamagnetism and photostability could realize cancer cells from a small amount of blood samples synchronous labeling and separation in a short time, which avoids tedious procedures of pretreatment of blood samples and purification of cells. Most of important, Owing to the interaction between biotin and streptavidin, the method based on biotin-labeled aptamer and FITC@Fe3O4@SiNPs-SA could be served as a universal bioprobe by simply changing the aptamer sequences for other targets detection in complex biological system. 3.2. TEM characterization of FITC@Fe3O4@SiNPs-COOH The size and morphology information of synthesized FITC@Fe3O4@SiNPs-COOH were measured using a transmission electron microscope (TEM). As shown in Fig. 2, the core-shell NPs were uniform in shape with a diameter of 60 ± 5 nm (the size of NPs analyzed by the SIS image-processing software). In addition, Fe3O4 as core represents the dark background, which was distributed evenly in the core, and silica as shell represents the bright background. So we could agree that Fe3O4 could be enwrapped successfully inside the silica shell. 3.3. Magnetic and fluorescent characterization of FITC@Fe3O4@SiNPs-COOH Superparamagnetic nanomaterials could be aggregated immediately in the presence of external magnetic field and dispersed rapidly in solution after the removal of external magnetic field due to no hysteresis, which have been widely used for bioseparation. Hysteresis loop is the most commonly used method to characterize the superparamagnetism. Magnetization (M) increases as the external magnetic field (H) increases in the hysteresis loop. The value of M no longer changes until H increases to a certain value, and the corresponding M represents the saturation magnetization (Ms). In contrast, the value of M decreases as the value of H decreases. However, for the usual magnetic materials, the corresponding M will not be reduced to zero when the H is reduced to zero. That is to say, the remanent magnetization represents hysteresis. An opposite direction magnetic field called coercive force, which could reduce the remanent magnetization to zero. For the superparamagnetic nanomaterials, as shown in Fig. 3A, the value of Ms is 21.6emu/g. In addition, the curve gets through the origin and shows the relationship between M and H in the range of 10 kOe ~10 kOe, which represents that the coercive force is zero. So we could agree that the prepared FITC@Fe3O4@SiNPs-COOH is
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Fig. 1. Overall schematic of the strategy based on aptamer and fluorescence-magnetic silica nanoprobes for MCF-7 cells synchronous labeling and separation.
superparamagnetic at room temperature. The fluorescence spectrum of FITC@Fe3O4@SiNPs-COOH was characterized by a fluorometer. As shown in Fig. 3B, compared with the fluorescence spectrum of free FITC dyes, FITC@Fe3O4@SiNPsCOOH have the same fluorescence emission wavelength (525 nm) with the excitation wavelength at 490 nm. Due to the mesoporous structure of silica, the FITC dyes entrapped inside the silica matrix are still excited. In addition, the prepared streptavidin-conjugated fluorescence-magnetic silica nanoprobe (FITC@Fe3O4@SiNPs-SA) was performed by XRD. From the XRD pattern (Supporting information, S1), the diffraction of the (220), (311), (400), (422), (511), and (440) planes may result in the corresponding diffraction peaks located at 2q of 29.7, 35.7, 43.4, 53.4, 57.4 and 62.7, respectively, which indicates the structure of Fe3O4. In addition, a distinct Wide dispersion peak located at 2q of 23 indicates that the silica spheres were amorphous crystal. 3.4. Application of the biotin-labeled aptamer and FITC@Fe3O4@SiNPs-SA for cancer cells synchronous labeling and separation Fig. 2. TEM image of carboxyl-modified fluorescence-magnetic silica nanoparticles (FITC@Fe3O4@SiNPs-COOH).
To investigate whether the method based on aptamer and
Fig. 3. (A) Magnetic hysteresis loop of the prepared carboxyl-modified fluorescence-magnetic silica nanoparticles (FITC@Fe3O4@SiNPs-COOH). (B) Fluorescence emission spectra of carboxyl-modified fluorescence-magnetic silica nanoparticles (FITC@Fe3O4@SiNPs-COOH).
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fluorescence-magnetic nanoprobe was practicable for cancer cells synchronous labeling and separation, MCF-7 cells suspension was orderly treated with biotin-labeled MUC-1 aptamer and FITC@Fe3O4@SiNPs-SA. After incubated, the cell-probe complexes were separated by a magnet, and then placed on a slide for fluorescence microscopic imaging. As shown in Fig. 4, A and B represent the bright-field and fluorescent microscopy pictures, respectively. From the picture A, cells still maintain good integrity and dispersity after separated by a magnet. Due to no hysteresis, the cells captured by FITC@Fe3O4@SiNPs-SA with superparamagnetism dispersed rapidly in solution after the removal of external magnetic field, which eliminates cells aggregation and mechanical damage caused by centrifugation. The corresponding cells with strong green fluorescence were clearly observed in the picture B. So we could agree that a large number of FITC@Fe3O4@SiNPs-SA were bound to the surface of each cell. To further demonstrate without nonspecific adsorption between cells and nanoprobes, and the high affinity and specificity of aptamer to targeted cell, the control groups were performed by orderly adding biotin-labeled random ssDNA and FITC@Fe3O4@SiNPs-SA to MCF-7 cells suspension. C and D represent the bright-field and fluorescent microscopy pictures in the control group, respectively. According to the scale bar, no cells were observed in both pictures C and D. in addition, the picture D only shows the green fluorescence of monodisperse FITC@Fe3O4@SiNPsSA. The biotin-labeled random ssDNA could not recognize and bind to the MUC-1 protein resulting in no biotin molecules on the surface of MCF-7 cells. Owing to the interaction between biotin and streptavidin, subsequent FITC@Fe3O4@SiNPs-SA would fail to capture the targeted cells. If without nonspecific adsorption between MCF-7 cells and nanoprobes, only single FITC@Fe3O4@SiNPs-SA were separated by a magnet. Then we believe that the MUC1aptamer could recognize and bind to MCF-7 cell with high
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affinity and specificity, and the prepared FITC@Fe3O4@SiNPs-SA could not nonspecifically bind to the MCF-7 cells. 3.5. Anti-photobleaching property of FITC@Fe3O4@SiNPs-SA As we know, photobleaching is the most serious problem for fluorescent dyes. To demonstrate the anti-photobleaching property of prepared nanoprobes, MCF-7 cells treated directly with FITClabeled MUC-1 aptamer, and treated orderly with biotin-labeled MUC-1 aptamer and FITC@Fe3O4@SiNPs-SA were excited under the same condition simultaneously. As shown in Fig. 5A, the fluorescent images were captured at 0 s, 60 s, 2 min, 5 min, and 10 min, respectively. According to the observation of naked eye, the green fluorescence of cells surface caused by FITC@Fe3O4@SiNPs-SA hardly changes even after successive intense irradiation for 10 min. While the fluorescence of cells surface caused by bare FITC was bleached quickly for 60 s irradiation, and bleached completely for 5 min irradiation. In addition, quantification of cell staining was performed using ImageJ software according to its standard manual and the reported method [32,33]. As shown in Fig. 5B, the fluorescence intensity of cells stained by both FITC@Fe3O4@SiNPs-SA and FITC dyes are consistent with the observation of naked eye. Therefore, the photostability of FITC@Fe3O4@SiNPs-SA is obviously superior to the pure FITC dye. The reason is that FITC molecules enwrapped in the silica shell may prevent FITC from oxidation due to protective effect of the silica shell [34,35]. 3.6. Application of the biotin-labeled aptamer and FITC@Fe3O4@SiNPs-SA for cancer cells synchronous labeling and separation in peripheral blood To further confirm whether the method based on aptamer and
Fig. 4. Fluorescence images of MCF-7 cells treated with the biotin-labeled MUC-1 aptamer and streptavidin-conjugated fluorescence-magnetic silica nanoprobes (FITC@Fe3O4@SiNPs-SA) (A, B) and treated with the biotin-labeled random ssDNA and FITC@Fe3O4@SiNPs-SA (C, D). A and B represent the bright-field and fluorescent microscopy pictures in the experimental group, respectively. C and D represent the bright-field and fluorescent microscopy pictures in the control group, respectively.
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Fig. 5. (A) Photostability comparison of FITC@Fe3O4@SiNPs-SA and FITC dye. MCF-7 cells were treated with the biotin-labeled MUC-1 aptamer and FITC@Fe3O4@SiNPs-SA (a1ea4), and FITC-labeled aptamer (b1eb4), respectively, and excited for 10 min by the successive intense irradiation. The fluorescence images were acquired at 0 s (a1 and b1), 60 s (a2 and b2), 5 min (a3 and b3), and 10 min (a4 and b4), respectively. Scale bar ¼ 25 mm. (B) Quantitative analysis of cancer cells, mean ± error bar (set at P < 0.05) versus different probes.
fluorescence-magnetic nanoprobe was practicable for cancer cells synchronous labeling and separation in complex biological system, the practical sample was performed by adding MCF-7 cells to the normal human peripheral blood treated with anticoagulant. The biotin-labeled MUC-1 aptamer was firstly added to the blood sample containing targeted cells. After incubation, centrifugation, and the removal of the upper solution, FITC@Fe3O4@SiNPs-SA was immediately added to the residual suspension. After incubated, the cell-probe complexes were separated by a magnet, and then placed on a slide for fluorescence microscopic imaging. As shown in Fig. 6, A and B represent the bright-field and fluorescent microscopy pictures, respectively. From the picture A, the cells with relatively vague morphology show good integrity and monodispersity. In addition, the corresponding cells with strong green fluorescence were clearly distinguished in the picture B. The aptamer binds specially to MCF-7 cells resulting in a larger number of negative charges on the cell surface. The electrostatic repulsion between MCF-7 cells and other cells especially for erythrocytes with negative charges in the blood facilitates the subsequent labeling and separation of FITC@Fe3O4@SiNPs-SA. However, nonspecific adsorption between some substances with positive charges and
targeted cells is unavoidable. C and D represent the bright-field and fluorescent microscopy pictures in the control group, respectively. No cells were observed either in picture C or picture D. Unlike the biotin-labeled MUC-1 aptamer, the biotin-labeled random ssDNA as a control experiment could not form special three-dimensional structure to recognize the MCF-7 cells as well as other cells in the blood sample. In addition, the repulsion of negative charges greatly eliminates the nonspecific adsorption between biotin-labeled random ssDNA and blood cells such as leukocyte, erythrocyte, and blood platelet. Moreover, after the blood samples were treated with the biotin-labeled random ssDNA, the unbound aptamer was removed by low-speed centrifugation and washing. Considering the above reasons, the biotin-labeled random ssDNA could not bind to any cells in the blood sample, which results in the impossibility of cells recognition and separation by adding FITC@Fe3O4@SiNPsSA. Moreover, owing to the properties of ignorable charge and large specific surface, FITC@Fe3O4@SiNPs-SA may eliminate the nonspecific binding to the pure MCF-7 cells as well as other cells such as leukocyte, erythrocyte, and blood platelet in the blood sample. So the picture D only shows dispersed green fluorescence of individual FITC@[email protected], we agree that the
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Fig. 6. MCF-7 cells synchronous labeling and separation in peripheral blood sample. The sample treated with the biotin-labeled MUC-1 aptamer and FITC@Fe3O4@SiNPs-SA (A, B), and treated with the biotin-labeled random ssDNA and FITC@Fe3O4@SiNPs-SA (C, D). A and B represent the bright-field and fluorescent microscopy pictures, respectively. C and D represent the bright-field and fluorescent microscopy pictures in the control group, respectively.
method is also applicable for cancer cells synchronous labeling and separation in complex biological system.
Conflicts of interest The authors declare that they have no conflict of interest.
4. Conclusions A simple and effective method based on biotin-labeled aptamer and streptavidin-conjugated fluorescence-magnetic silica nanoprobes (FITC@Fe3O4@SiNPs-SA) for human breast carcinoma MCF7 cells synchronous labeling and separation had been established. The MUC-1 aptamer shows high selectivity and affinity to MCF7 cells. FITC@Fe3O4@SiNPs-SA eliminates the nonspecific binding to the pure MCF-7 cells as well as other cells such as leukocyte, erythrocyte, and blood platelet in the blood sample because of its ignorable charge and large specific surface, which could be applied effectively for MCF-7 cells synchronous labeling and separation in both suspension and blood sample. In addition, FITC@Fe3O4@SiNPs-SA takes advantage of photostable property and superparamagnetism, which displays anti-photobleaching property than the dye-labeled probes, and eliminates cells aggregation and mechanical damage caused by centrifugation. Moreover, silica nanomaterials with good biocompatibility, which could be served as a necessary and useful supplement for cancer cells ultrasensitive detection in various complex backgrounds.
Compliance with ethical standards The blood sample was acquired with the permission of the donor, and all the research procedures were approved by the Ethics Committee of Wuxi school of medicine, Jiangnan University.
Acknowledgments This work was financially supported by the fund of the development of science and technology of Wuxi (No. CSE31N626) and the scientific fund from Health and family planning commission of Wuxi (No. Q201627). Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.optmat.2017.11.003. References [1] M. Lin, J.F. Chen, Y.T. Lu, Y. Zhang, J.Z. Song, S. Hou, Z. Ke, H.R. Tseng, Nanostructure embedded microchips for detection, isolation, and characterization of circulating tumor cells, Acc. Chem. Res. 47 (2014) 2941e2950. [2] J.J. Salomon, V.E. Muchitsch, J.C. Gausterer, E. Schwagerus, H. Huwer, N. Daum, C.M. Lehr, C. Ehrhardt, The cell line NCl-H441 is a useful in vitro model for transport studies of human distal lung epithelial barrier, Mol. Pharm. 11 (2014) 995e1006. [3] J. Park, M.K. Ha, N. Yang, T.H. Yoon, Flow cytometry-based quantification of cellular Au nanoparticles, Anal. Chem. 89 (2017) 2449e2456. [4] S. Kim, S.I. Han, M.J. Park, C.W. Jeon, Y.D. Joo, I.H. Choi, I.H. Choi, K.H. Han, Circulating tumor cell microseparator based on lateral magnetophoresis and immunomagnetic nanobeads, Anal. Chem. 85 (2013) 2779e2786. [5] Y.H. Zeng, D.F. Kelley, Two-photon photochemistry of CdSe quantum dots, ACS Nano 9 (2015) 10471e10481. [6] S.T. Yang, L. Cao, P.G. Luo, F.S. Lu, X. Wang, H.F. Wang, M.J. Meziani, Y.F. Liu, G. Qi, Y.P. Sun, Carbon dots for optical imaging in vivo, J. Am. Chem. Soc. 131
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Optical Materials journal homepage: www.elsevier.com/locate/optmat
Breast cancer cells synchronous labeling and separation based on aptamer and fluorescence-magnetic silica nanoparticles Qiu-Yue Wang a, Wei Huang b, Xing-Lin Jiang a, Yan-Jun Kang b, * a b
Hunan University of Medicine, Huaihua, Hunan 418000, PR China Wuxi School of Medicine, Jiangnan University, Wuxi, Jiangsu 214122, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 July 2017 Received in revised form 28 October 2017 Accepted 3 November 2017
In this work, an efficient method based on biotin-labeled aptamer and streptavidin-conjugated fluorescence-magnetic silica nanoprobes (FITC@Fe3O4@SiNPs-SA) has been established for human breast carcinoma MCF-7 cells synchronous labeling and separation. Carboxyl-modified fluorescence-magnetic € ber method. Strepsilica nanoparticles (FITC@Fe3O4@SiNPs-COOH) were first synthesized using the Sto tavidin (SA) was then conjugated to the surface of FITC@Fe3O4@SiNPs-COOH. The MCF-7 cell suspension was incubated with biotin-labeled MUC-1 aptamer. After centrifugation and washing, the cells were then treated with FITC@Fe3O4@SiNPs-SA. Afterwards, the mixtures were separated by a magnet. The cellprobe conjugates were then imaged using fluorescent microscopy. The results show that the MUC-1 aptamer could recognize and bind to the targeted cells with high affinity and specificity, indicating the prepared FITC@Fe3O4@SiNPs-SA with great photostability and superparamagnetism could be applied effectively in labeling and separation for MCF-7 cell in suspension synchronously. In addition, the feasibility of MCF-7 cells detection in peripheral blood was assessed. The results indicate that the method above is also applicable for cancer cells synchronous labeling and separation in complex biological system. © 2017 Elsevier B.V. All rights reserved.
Keywords: Fluorescence Magnetism Silica nanoprobes Aptamer Streptavidin MCF-7 cells
1. Introduction Breast cancer is one of the commonest malignant tumors in women. According to the statistics, there are about 1.2 million new patients with breast cancer each year in the world, of which 0.5 million patients died of breast cancer. Tumor metastasis is the leading cause of death in patients. Circulating tumor cell (CTC) is a kind of tumor cell that exists in peripheral blood, which spreads to other organs of the body by the blood circulation [1]. However, most of CTCs in peripheral blood suffer from apoptosis or cytophagy. Very few of CTCs escape and develop into metastatic lesion, and increase the mortality risk of patients. Therefore, the early detection of breast cancer CTC in the blood would play important guiding roles in the terms of monitoring of tumor recurrence, therapeutic evaluation, and individual treatment. At present, CTCs are mainly detected using immunocytochemistry (ICC), Flow cytometry (FCM), and RT-PCR [2e4]. Briefly, ICC reflects the existence and distribution of biomacromolecules such
* Corresponding author. E-mail address: [email protected] (Y.-J. Kang). https://doi.org/10.1016/j.optmat.2017.11.003 0925-3467/© 2017 Elsevier B.V. All rights reserved.
as proteins, membrane surface antigens, and receptors, but the procedure is tedious and time-consuming. In addition, the poor photostability of fluorescein may influence the observation results. Both FCM and RT-PCR are sensitive and fast, which can quantitatively analyze the cell components such as DNA. However, cell morphology and more biological information can't be acquired by the above methods. How to develop an efficient and sensitive method for CTCs detection in complex biological system is essential in bioanalysis. With the development of nanotechnology in recent years, some fluorescent nanomaterials such as quantum dots, carbon dots, and dye-doped silica nanoparticles have showed great advantages in the field of biomedical imaging because of their unique optical properties [5e7]. In addition, the AgInZnS nanoparticles used for cell labeling is also reported [8]. However, faced with the complex system, these materials only play a role of single labeling. Other substances in the mixtures may reduce the labeling efficiency of targets and disturb the signals of detection. To enhance the sensitivity, the process of pretreatment and purification is necessary for most fluorescent-labeling based methods. In addition, although magnetic nanomaterials such as a-Fe, Fe3O4, and aFe2O3 have aroused great concern in the field of separation science
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[9e11], they mainly provide single separation. Besides, some magnetic materials are prone to aggregation after the removal of external magnetic field due to the hysteresis, which results in poor separation of targets. Moreover, the complex procedure of preparing biomolecules-functionalized magnetic nanoprobes and poor biocompatibility of bare magnetic nanomaterials should be considered [12]. Therefore, it is urgent to prepare composite nanomaterials with fluorescence, superparamagnetism, and good biocompatibility for targets synchronous labeling and separation. Composite silica nanomaterials have attracted considerable attention in biomedical analysis because of their antiphotobleaching, larger specific surface area, small size, good biocompatibility, facile surface modification, and special magnetic properties [13e15]. Especially, dye-doped silica nanoparticles have been widely used for cancer cells labeling, DNA microanalysis, and the detection of pathogenic bacteria [16e18]. In addition, Fe3O4enwrapped silica nanoparticles (Fe3O4@SiO2) with superparamagnetism could be aggregated immediately in the presence of external magnetic field and dispersed rapidly in solution after the removal of external magnetic field due to no hysteresis, which have been widely used for separation, and magnetic resonance imaging (MRI) [19e21]. In this paper, to prepare composite silica nanoprobes with fluorescence and magnetism for targets synchronous labeling and separation, Fe3O4 nanoparticles and fluorescent dyes (FITC) are enwrapped simultaneously into the silica € ber method [22,23]. Targeted nanoprobes networks using the Sto are then prepared by conjugating the recognizable biomolecules to the surface of fluorescence-magnetic silica nanoparticles. As we know, antibodies as the conventional recognition elements have been widely used for biolabeling. However, they can't meet the demands of modern biological analysis increasingly. Exploring promising recognition elements has always been a goal pursued by researchers. In recent years, the aptamer based on the systematic evolution of ligands by exponential enrichment (SELEX) has attracted great concern [24]. Aptamers are single-stranded nucleic with three-dimensional structure, which show high affinity and specificity with all kinds of targets such as cancer cells, pathogenic bacteria, Virus, ATP, and even metal ions [25e29]. Compared with antibodies, aptamers take advantages of strong tissue permeability, ignorable immunogenicity, high affinity and specificity with flexible targets, easy preparation and preservation, and small molecular weight. Inspired by the extraordinary properties of aptamer and fluorescence-magnetic silica nanoparticles, we develop an efficient and sensitive method based on biotinlabeled aptamer and streptavidin-conjugated fluorescence-magnetic silica nanoprobes for breast cancer cells in the blood synchronous labeling and separation. This technology not only sensitively, conveniently, real-timely, and dynamically indicates the tumor progression and molecular biology information but also plays important guiding roles in the terms of monitoring of tumor recurrence, therapeutic evaluation, and individual treatment. 2. Experimental 2.1. Chemicals and materials TritonX-100, tetraethyl orthosilicate (TEOS), (3-aminopropyl) triethoxysilane (APTES), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimid ehydrochloride (EDC), N-hydroxysulfosuccinimide sodium salt (sulfo-NHS), Fluorescein isothiocyanate (FITC), and Streptavidin were purchased from Sigma Chemical Co. (St. Louis. MO). Ammonium hydroxide (25-28 wt%), Ferric chloride hexahydrate (FeCl3$6H2O), Ferrous chloride tetrahydrate (FeCl2$4H2O), alcohol, N,-N-Dimethylformamide (DMF), and Succinic anhydride were purchased from China Pharmaceutical Group Shanghai
Chemical Reagent Co., Ltd. Human breast carcinoma MCF-7 cells lines were obtained from china center for type culture collection. FITC-labeled MUC-1 aptamer was synthesized from Beijing Biosynthesis Biotechnology Co.,Ltd. The biotin-labeled MUC-1 aptamer, and the biotin-labeled random ssDNA were synthesized in Shanghai Sangon Biological Engineering Technology & Services Co. (China), and the sequence as follows: 5/-biotin-(CH2)6-GGGAGACAAGAATAAACGCTCAAGCAGTTGATC CTTTGGATACCCTGGTTCGACAGGAGGCTCACAACAGGC-3/ 5/-biotin-(CH2)6-ACAGGAGGCTCACAAACGCTCAAACAGGAGGCT CACTAACGGACGTTCGACAGGAGGCTGGACGTTCGACGA-3/ 2.2. Main instrumentation The size and uniformity of synthesized nanoparticles were measured by means of transmission electron microscope (JEOL, JEM100CXII, Japan). Photoluminescence was measured using fluorescence spectrophotometer (F96PRO, China). Fluorescence images results were observed with under inverted fluorescence microscope (Nikon ECLIPSE TE2000-U, Japan). Magnetic characterization of synthesized nanoparticles was performed using vibrating sample magnetometer (Lakeshore 7410, USA). Structural analysis of prepared nanoprobe was performed using X-ray diffraction (XRD6000, SHIMADZU). 2.3. Fe3O4 nanoparticle synthesis Fe3O4 nanoparticles were prepared by the coprecipitation method. Briefly, 6.76 g of FeCl3$6H2O, 2.49 g of FeCl2$4H2O, and 200 mL of deionized water were added together to a flask. 0.4 M ammonia hydroxide was then gradually added to the iron chloride solution stirred at 350 rpm using a mechanical stirrer until pH reached 10.0. The reaction was allowed to continue for 30 min at 30 C, followed by centrifuging and washing with deionized water three times. The products were finally resuspended in 95% ethanol for the next step. 2.4. Preparation of FITC-APTES precursor 35 mL of APTES and 3 mg of FITC were mixed in 0.5 mL of absolute ethanol under dry nitrogen atmosphere and stirring magnetically for 12 h. In addition, the FITC-APTES conjugates solution was protected from light during reaction and storage. 2.5. Preparation of carboxyl-modified fluorescent-magnetic silica nanoparticles Carboxyl-modified fluorescence-magnetic silica nanoparticles € ber (FITC@Fe3O4@SiNPs-COOH) were synthesized using the Sto method. Briefly, 40 mL of ethanol, 10 mL of deionized water, and 0.5 mL of 50 mg/mL Fe3O4 were sonicated for 90 min and then stirred at 350 rpm using a mechanical stirrer. 1 mL of ammonia hydroxide and 200 mL of FITC-APTES were then added to the solution stirred. To create the silica coating, 1 mL of TEOS was added, and the mixture was stirred for 4 h to complete the hydrolysis process. For postcoating, 50 mL of TEOS and 50 mL of APTES were added and additional stir was performed for 2 h. By washing and resuspending with DMF solution, the products were reacted with succinic anhydride under nitrogen gas for 24 h with continuous stirring. Then the prepared FITC@Fe3O4@SiNPs-COOH were washed by water and resuspended in PBS for the next step. 2.6. Conjugation of streptavidin onto FITC@Fe3O4@SiNPs-COOH 0.5 mL of FITC@Fe3O4@SiNPs-COOH, 1 mg of EDC, and 2.5 mg of
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Sulfo-NHS were added to 2 mL of 0.1 M PBS buffer (pH 7.4). The mixture was then incubated for 15 min at room temperature with gentle shaking. After the reaction completed, 50 mL of 1 mg/mL streptavidin was immediately added to the solution, following by 3 h incubation with gentle shaking at room temperature. Particles were washed with 0.1 M PBS (pH 7.4) and then resuspended in 1 ml of 0.05% BSA for 1 h to block the free carboxylates. After the reaction completed, the prepared streptavidin-conjugated fluorescencemagnetic silica nanoparticles (FITC@Fe3O4@SiNPs-SA) were washed with 0.1 M PBS buffer (pH 7.4) three times and then resuspended in 0.1 M PBS (pH 7.4) for further use. 2.7. Cancer cells synchronous labeling and separation Human breast carcinoma MCF-7 cells were cultured until the cover rate on the six-well plate reached 70e90%. After the removal of the medium, the adherent cells were digested by pancreatin. Fresh medium was then added to the six-well plate, and the suspended cells were centrifuged (1000 rpm 5 min). After the removal of the supernatant, the collected cells were resuspended in 2 mL of 0.1 M PBS (pH 7.4). 100 mL of cell suspension was diluted 10fold with PBS. 100 mL of 10 mM biotin-labeled MUC-1 aptamer and cells suspension diluted were incubated for 60 min at 37 C with gentle shaking. After the reaction completed, the suspension of cells-aptamer complexes were centrifuged and washed with PBS three times, and then resuspended in 1 mL of 0.1 M PBS (pH 7.4). 50 mL of FITC@Fe3O4@SiNPs-SA was immediately added to the suspension. After incubated for 60 min at 37 C with gentle shaking, the mixtures were separated by a magnet and washed with 0.1 M PBS (pH 7.4) three times. The cell-nanoprobe conjugates were finally resuspended in PBS and then placed on the slide glass for fluorescence microscope observation. Corresponding control group was treated through the same procedure with exception of the biotin-labeled MUC-1 aptamer was replaced by biotin-labeled random ssDNA. 2.8. Cancer cells synchronous labeling and separation in peripheral blood 100 mL of MCF-7 cells in 0.1 M PBS (pH 7.4) with a concentration of 103 cell/mL was added to 1.5 mL of normal human peripheral blood sample in the presence of 2 mg of EDTA dipotassium salt. After mixed fully, 100 mL of 10 mM biotin-labeled MUC-1 aptamer was then added to the blood sample above. The samples were incubated for 60 min at 37 C with gentle shaking, and then centrifuged (4000 rpm 5 min). After the removal of the upper solution, 100 mL of FITC@Fe3O4@SiNPs-SA was immediately added to the residual suspension. After incubated for 60 min at 37 C with gentle shaking, the suspension was treated by a magnet and washed with 0.1 M PBS (pH 7.4) three times. The acquired products were resuspended in PBS, and finally placed on a slide for fluorescence microscopic imaging. The control groups follow the steps above except that the biotin-labeled random ssDNA was used instead of biotin-labeled MUC-1 aptamer. 3. Results and discussion 3.1. Strategy based on aptamer and fluorescence-magnetic silica nanoprobes for MCF-7 cells synchronous labeling and separation In this paper, we used the reported aptamer with 72 bases as the recognition element because of its high affinity and specificity to the MUC-1 protein on the surface of human breast carcinoma MCF7 cells [30,31]. To avoid the steric hindrance between cells and nanoprobes, the biotin-labeled aptamer and streptavidin-
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conjugated fluorescence-magnetic nanoprobe (FITC@Fe3O4@SiNPs-SA) were individually used instead of aptamer directly conjugated nanoprobes (FITC@Fe3O4@SiNPs-aptamer). The overall schematic is described in Fig. 1. The mixtures containing MCF7 cells were firstly treated with the free biotin-labeled MUC-1 aptamer. After incubated, the unbound biotin-labeled aptamer was removed by low-speed centrifugation and washing. Owing to the interaction between biotin and streptavidin, the targeted cells were subsequently captured by adding FITC@Fe3O4@SiNPs-SA to the resuspended cells. After incubated, the cell-probe complexes were separated by a magnet, and then placed on a slide for fluorescence microscopic imaging. The control groups follow the steps above except that the biotin-labeled random ssDNA was used instead of biotin-labeled MUC-1 aptamer. First of all, the aptamer as the promising recognition element was used instead of antibodies because of its high affinity and specificity with flexible targets, easy preparation and preservation, small molecular weight, strong tissue permeability, and ignorable immunogenicity. Moreover, compared with the methods of single separation and labeling, the fluorescence-magnetic silica nanoprobes (FITC@Fe3O4@SiNPs-SA) with superparamagnetism and photostability could realize cancer cells from a small amount of blood samples synchronous labeling and separation in a short time, which avoids tedious procedures of pretreatment of blood samples and purification of cells. Most of important, Owing to the interaction between biotin and streptavidin, the method based on biotin-labeled aptamer and FITC@Fe3O4@SiNPs-SA could be served as a universal bioprobe by simply changing the aptamer sequences for other targets detection in complex biological system. 3.2. TEM characterization of FITC@Fe3O4@SiNPs-COOH The size and morphology information of synthesized FITC@Fe3O4@SiNPs-COOH were measured using a transmission electron microscope (TEM). As shown in Fig. 2, the core-shell NPs were uniform in shape with a diameter of 60 ± 5 nm (the size of NPs analyzed by the SIS image-processing software). In addition, Fe3O4 as core represents the dark background, which was distributed evenly in the core, and silica as shell represents the bright background. So we could agree that Fe3O4 could be enwrapped successfully inside the silica shell. 3.3. Magnetic and fluorescent characterization of FITC@Fe3O4@SiNPs-COOH Superparamagnetic nanomaterials could be aggregated immediately in the presence of external magnetic field and dispersed rapidly in solution after the removal of external magnetic field due to no hysteresis, which have been widely used for bioseparation. Hysteresis loop is the most commonly used method to characterize the superparamagnetism. Magnetization (M) increases as the external magnetic field (H) increases in the hysteresis loop. The value of M no longer changes until H increases to a certain value, and the corresponding M represents the saturation magnetization (Ms). In contrast, the value of M decreases as the value of H decreases. However, for the usual magnetic materials, the corresponding M will not be reduced to zero when the H is reduced to zero. That is to say, the remanent magnetization represents hysteresis. An opposite direction magnetic field called coercive force, which could reduce the remanent magnetization to zero. For the superparamagnetic nanomaterials, as shown in Fig. 3A, the value of Ms is 21.6emu/g. In addition, the curve gets through the origin and shows the relationship between M and H in the range of 10 kOe ~10 kOe, which represents that the coercive force is zero. So we could agree that the prepared FITC@Fe3O4@SiNPs-COOH is
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Fig. 1. Overall schematic of the strategy based on aptamer and fluorescence-magnetic silica nanoprobes for MCF-7 cells synchronous labeling and separation.
superparamagnetic at room temperature. The fluorescence spectrum of FITC@Fe3O4@SiNPs-COOH was characterized by a fluorometer. As shown in Fig. 3B, compared with the fluorescence spectrum of free FITC dyes, FITC@Fe3O4@SiNPsCOOH have the same fluorescence emission wavelength (525 nm) with the excitation wavelength at 490 nm. Due to the mesoporous structure of silica, the FITC dyes entrapped inside the silica matrix are still excited. In addition, the prepared streptavidin-conjugated fluorescence-magnetic silica nanoprobe (FITC@Fe3O4@SiNPs-SA) was performed by XRD. From the XRD pattern (Supporting information, S1), the diffraction of the (220), (311), (400), (422), (511), and (440) planes may result in the corresponding diffraction peaks located at 2q of 29.7, 35.7, 43.4, 53.4, 57.4 and 62.7, respectively, which indicates the structure of Fe3O4. In addition, a distinct Wide dispersion peak located at 2q of 23 indicates that the silica spheres were amorphous crystal. 3.4. Application of the biotin-labeled aptamer and FITC@Fe3O4@SiNPs-SA for cancer cells synchronous labeling and separation Fig. 2. TEM image of carboxyl-modified fluorescence-magnetic silica nanoparticles (FITC@Fe3O4@SiNPs-COOH).
To investigate whether the method based on aptamer and
Fig. 3. (A) Magnetic hysteresis loop of the prepared carboxyl-modified fluorescence-magnetic silica nanoparticles (FITC@Fe3O4@SiNPs-COOH). (B) Fluorescence emission spectra of carboxyl-modified fluorescence-magnetic silica nanoparticles (FITC@Fe3O4@SiNPs-COOH).
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fluorescence-magnetic nanoprobe was practicable for cancer cells synchronous labeling and separation, MCF-7 cells suspension was orderly treated with biotin-labeled MUC-1 aptamer and FITC@Fe3O4@SiNPs-SA. After incubated, the cell-probe complexes were separated by a magnet, and then placed on a slide for fluorescence microscopic imaging. As shown in Fig. 4, A and B represent the bright-field and fluorescent microscopy pictures, respectively. From the picture A, cells still maintain good integrity and dispersity after separated by a magnet. Due to no hysteresis, the cells captured by FITC@Fe3O4@SiNPs-SA with superparamagnetism dispersed rapidly in solution after the removal of external magnetic field, which eliminates cells aggregation and mechanical damage caused by centrifugation. The corresponding cells with strong green fluorescence were clearly observed in the picture B. So we could agree that a large number of FITC@Fe3O4@SiNPs-SA were bound to the surface of each cell. To further demonstrate without nonspecific adsorption between cells and nanoprobes, and the high affinity and specificity of aptamer to targeted cell, the control groups were performed by orderly adding biotin-labeled random ssDNA and FITC@Fe3O4@SiNPs-SA to MCF-7 cells suspension. C and D represent the bright-field and fluorescent microscopy pictures in the control group, respectively. According to the scale bar, no cells were observed in both pictures C and D. in addition, the picture D only shows the green fluorescence of monodisperse FITC@Fe3O4@SiNPsSA. The biotin-labeled random ssDNA could not recognize and bind to the MUC-1 protein resulting in no biotin molecules on the surface of MCF-7 cells. Owing to the interaction between biotin and streptavidin, subsequent FITC@Fe3O4@SiNPs-SA would fail to capture the targeted cells. If without nonspecific adsorption between MCF-7 cells and nanoprobes, only single FITC@Fe3O4@SiNPs-SA were separated by a magnet. Then we believe that the MUC1aptamer could recognize and bind to MCF-7 cell with high
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affinity and specificity, and the prepared FITC@Fe3O4@SiNPs-SA could not nonspecifically bind to the MCF-7 cells. 3.5. Anti-photobleaching property of FITC@Fe3O4@SiNPs-SA As we know, photobleaching is the most serious problem for fluorescent dyes. To demonstrate the anti-photobleaching property of prepared nanoprobes, MCF-7 cells treated directly with FITClabeled MUC-1 aptamer, and treated orderly with biotin-labeled MUC-1 aptamer and FITC@Fe3O4@SiNPs-SA were excited under the same condition simultaneously. As shown in Fig. 5A, the fluorescent images were captured at 0 s, 60 s, 2 min, 5 min, and 10 min, respectively. According to the observation of naked eye, the green fluorescence of cells surface caused by FITC@Fe3O4@SiNPs-SA hardly changes even after successive intense irradiation for 10 min. While the fluorescence of cells surface caused by bare FITC was bleached quickly for 60 s irradiation, and bleached completely for 5 min irradiation. In addition, quantification of cell staining was performed using ImageJ software according to its standard manual and the reported method [32,33]. As shown in Fig. 5B, the fluorescence intensity of cells stained by both FITC@Fe3O4@SiNPs-SA and FITC dyes are consistent with the observation of naked eye. Therefore, the photostability of FITC@Fe3O4@SiNPs-SA is obviously superior to the pure FITC dye. The reason is that FITC molecules enwrapped in the silica shell may prevent FITC from oxidation due to protective effect of the silica shell [34,35]. 3.6. Application of the biotin-labeled aptamer and FITC@Fe3O4@SiNPs-SA for cancer cells synchronous labeling and separation in peripheral blood To further confirm whether the method based on aptamer and
Fig. 4. Fluorescence images of MCF-7 cells treated with the biotin-labeled MUC-1 aptamer and streptavidin-conjugated fluorescence-magnetic silica nanoprobes (FITC@Fe3O4@SiNPs-SA) (A, B) and treated with the biotin-labeled random ssDNA and FITC@Fe3O4@SiNPs-SA (C, D). A and B represent the bright-field and fluorescent microscopy pictures in the experimental group, respectively. C and D represent the bright-field and fluorescent microscopy pictures in the control group, respectively.
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Fig. 5. (A) Photostability comparison of FITC@Fe3O4@SiNPs-SA and FITC dye. MCF-7 cells were treated with the biotin-labeled MUC-1 aptamer and FITC@Fe3O4@SiNPs-SA (a1ea4), and FITC-labeled aptamer (b1eb4), respectively, and excited for 10 min by the successive intense irradiation. The fluorescence images were acquired at 0 s (a1 and b1), 60 s (a2 and b2), 5 min (a3 and b3), and 10 min (a4 and b4), respectively. Scale bar ¼ 25 mm. (B) Quantitative analysis of cancer cells, mean ± error bar (set at P < 0.05) versus different probes.
fluorescence-magnetic nanoprobe was practicable for cancer cells synchronous labeling and separation in complex biological system, the practical sample was performed by adding MCF-7 cells to the normal human peripheral blood treated with anticoagulant. The biotin-labeled MUC-1 aptamer was firstly added to the blood sample containing targeted cells. After incubation, centrifugation, and the removal of the upper solution, FITC@Fe3O4@SiNPs-SA was immediately added to the residual suspension. After incubated, the cell-probe complexes were separated by a magnet, and then placed on a slide for fluorescence microscopic imaging. As shown in Fig. 6, A and B represent the bright-field and fluorescent microscopy pictures, respectively. From the picture A, the cells with relatively vague morphology show good integrity and monodispersity. In addition, the corresponding cells with strong green fluorescence were clearly distinguished in the picture B. The aptamer binds specially to MCF-7 cells resulting in a larger number of negative charges on the cell surface. The electrostatic repulsion between MCF-7 cells and other cells especially for erythrocytes with negative charges in the blood facilitates the subsequent labeling and separation of FITC@Fe3O4@SiNPs-SA. However, nonspecific adsorption between some substances with positive charges and
targeted cells is unavoidable. C and D represent the bright-field and fluorescent microscopy pictures in the control group, respectively. No cells were observed either in picture C or picture D. Unlike the biotin-labeled MUC-1 aptamer, the biotin-labeled random ssDNA as a control experiment could not form special three-dimensional structure to recognize the MCF-7 cells as well as other cells in the blood sample. In addition, the repulsion of negative charges greatly eliminates the nonspecific adsorption between biotin-labeled random ssDNA and blood cells such as leukocyte, erythrocyte, and blood platelet. Moreover, after the blood samples were treated with the biotin-labeled random ssDNA, the unbound aptamer was removed by low-speed centrifugation and washing. Considering the above reasons, the biotin-labeled random ssDNA could not bind to any cells in the blood sample, which results in the impossibility of cells recognition and separation by adding FITC@Fe3O4@SiNPsSA. Moreover, owing to the properties of ignorable charge and large specific surface, FITC@Fe3O4@SiNPs-SA may eliminate the nonspecific binding to the pure MCF-7 cells as well as other cells such as leukocyte, erythrocyte, and blood platelet in the blood sample. So the picture D only shows dispersed green fluorescence of individual FITC@[email protected], we agree that the
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Fig. 6. MCF-7 cells synchronous labeling and separation in peripheral blood sample. The sample treated with the biotin-labeled MUC-1 aptamer and FITC@Fe3O4@SiNPs-SA (A, B), and treated with the biotin-labeled random ssDNA and FITC@Fe3O4@SiNPs-SA (C, D). A and B represent the bright-field and fluorescent microscopy pictures, respectively. C and D represent the bright-field and fluorescent microscopy pictures in the control group, respectively.
method is also applicable for cancer cells synchronous labeling and separation in complex biological system.
Conflicts of interest The authors declare that they have no conflict of interest.
4. Conclusions A simple and effective method based on biotin-labeled aptamer and streptavidin-conjugated fluorescence-magnetic silica nanoprobes (FITC@Fe3O4@SiNPs-SA) for human breast carcinoma MCF7 cells synchronous labeling and separation had been established. The MUC-1 aptamer shows high selectivity and affinity to MCF7 cells. FITC@Fe3O4@SiNPs-SA eliminates the nonspecific binding to the pure MCF-7 cells as well as other cells such as leukocyte, erythrocyte, and blood platelet in the blood sample because of its ignorable charge and large specific surface, which could be applied effectively for MCF-7 cells synchronous labeling and separation in both suspension and blood sample. In addition, FITC@Fe3O4@SiNPs-SA takes advantage of photostable property and superparamagnetism, which displays anti-photobleaching property than the dye-labeled probes, and eliminates cells aggregation and mechanical damage caused by centrifugation. Moreover, silica nanomaterials with good biocompatibility, which could be served as a necessary and useful supplement for cancer cells ultrasensitive detection in various complex backgrounds.
Compliance with ethical standards The blood sample was acquired with the permission of the donor, and all the research procedures were approved by the Ethics Committee of Wuxi school of medicine, Jiangnan University.
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