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Clinical Chemistry 53: 2177-2185, 2007. First published October 25, 2007; 10.1373/clinchem.2007.092023
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(Clinical Chemistry. 2007;53:2177-2185.)
© 2007 American Association for Clinical Chemistry, Inc.

Automation and Analytical Techniques

Visual Recognition and Efficient Isolation of Apoptotic Cells with Fluorescent-Magnetic-Biotargeting Multifunctional Nanospheres

Er-Qun Song1, Guo-Ping Wang1, Hai-Yan Xie2, Zhi-Ling Zhang1, Jun Hu1, Jun Peng1, Dao-Cheng Wu3, Yun-Bo Shi4 and Dai-Wen Pang1,a

1 College of Chemistry and Molecular Sciences and State Key Laboratory of Virology, Wuhan University, Wuhan, Peoples Republic of China.
2 School of Life Science and Technology, Beijing Institute of Technology, Beijing, Peoples Republic of China.
3 School of Life Science and Technology, Xi’an Jiaotong University, Peoples Republic of China.
4 Section on Molecular Morphogenesis, Program on Cell Regulation and Metabolism, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD.

aAddress correspondence to this author at: College of Chemistry and Molecular Sciences and State Key Laboratory of Virology, Wuhan University, Wuhan 430072, Peoples Republic of China. Fax 86-27-6875-4067; e-mail dwpang{at}whu.edu.cn.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Background: Fluorescent-magnetic-biotargeting multifunctional nanospheres are likely to find important applications in bioanalysis, biomedicine, and clinical diagnosis. We have been developing such multifunctional nanospheres for biomedical applications.

Methods: We covalently coupled avidin onto the surfaces of fluorescent-magnetic bifunctional nanospheres to construct fluorescent-magnetic-biotargeting trifunctional nanospheres and analyzed the functionality and specificity of these trifunctional nanospheres for their ability to recognize and isolate apoptotic cells labeled with biotinylated annexin V, which recognizes phosphatidylserine exposed on the surfaces of apoptotic cells.

Results: The multifunctional nanospheres can be used in combination with propidium iodide staining of nuclear DNA to identify cells at different phases of the apoptotic process. Furthermore, we demonstrate that apoptotic cells induced by exposure to ultraviolet light can be isolated simply with a magnet from living cells at an efficiency of at least 80%; these cells can then be easily visualized with a fluorescence microscope.

Conclusions: Our results show that fluorescent-magnetic-biotargeting trifunctional nanospheres can be a powerful tool for rapidly recognizing, magnetically enriching and sorting, and simultaneously identifying different kinds of cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
Rapid recognition and sorting of cells are important in the developing fields of cellular therapy and biotechnology. The separation and analysis of different types of individual cells, such as cancer cells, T cells (the first line of defense against invading pathogens), and apoptotic cells associated with many kinds of diseases(1)(2)(3) are becoming increasingly important, not only for basic research but also, more importantly, for drug screening and development and disease diagnosis. Techniques for recognizing and separating specific target cells from a mixed-cell population are urgently needed for such biomedical analyses and clinical applications. Of the methods currently used to recognize and isolate types of individual cells for biochemical or functional studies, the most widely used are fluorescence-activated cell recognition techniques such as fluorescence imaging(4), flow cytometry(5)(6)(7), and magnetically activated cell sorting [e.g., immunomagnetic microsphere-based selection(8)(9)(10)]. In general, the sorting of cells is based on recognition, which requires sufficient random collisions between the cell-specific microspheres and the target cells. Evidently, it is difficult to get heavy microspheres to interact with micrometer-sized target cells; however, the use of nanospheres promises to solve such problems, because the lightness and small sizes of nanospheres facilitate their collision and interaction with larger objects such as cells.

Nanomaterials are increasingly being used in biology and medicine, particularly in the fields of cell recognition and separation. Cell-targeting microspheres conjugated with organic dyes have broad applications in recognizing and imaging target cells(11)(12). Quantum dots (QDs),1 relatively new colloidal semiconductor nanocrystals, have also attracted increasing attention in fluorescence applications, including cell imaging and recognition(13)(14), because of their unique optical properties(15)(16). In contrast with the use of organic dyes, the ease of generating QDs with different emission wavelengths facilitates the use of QDs for simultaneously recognizing and imaging several kinds of cells, and this recognition can be modified through the use of different biotargeting molecules. On the other hand, magnetic nanoparticles offer the capability of cell isolation from original or enriched samples without the use of centrifugation or filtration(17)(18)(19)(20)(21)(22). In 1990, Miltenyi et al.(23) developed a method for separating and identifying rare cells that uses magnetic nanoparticles coupled to an antibody tagged with a fluorescent dye; however, organic dyes have several disadvantageous properties that limit the use of this method. More recently, the combination of fluorescent QDs and magnetic nanoparticles into single nanospheres to obtain fluorescent-magnetic bifunctional nanospheres (FMBNs) has created the potential for broader applications in biomedicine and in clinical diagnosis. We previously synthesized such FMBNs by coembedding QDs and nano-{gamma}-Fe2O3 magnetic nanoparticles into swelling poly(styrene/acrylamide) nanospheres(24). In addition, several other approaches incorporating the use of QDs and magnetic nanoparticles have recently been applied to the synthesis of fluorescent-magnetic bifunctional nanoparticles(25)(26)(27)(28). More recently, we developed several types of biotargeting trifunctional nanospheres by coembedding QDs and nano-{gamma}-Fe2O3 particles into poly(styrene/acrylamide) copolymer nanospheres and then covalently coupling different biological reagents, such as folic acid, biotin, avidin, and antibody, onto the surfaces of the nanospheres(24)(29)(30)(31).

In this study, we used apoptosis as a model to investigate the potential of trifunctional nanospheres for visualizing and efficiently isolating specific cells. Apoptosis, a critical process in the development and homeostasis of multicellular organisms, has been associated with many diseases, including AIDS and tumor development(1)(2)(3). Our understanding of the progression of apoptosis and analyses of the associated changes in DNA, proteins, or organelles would greatly benefit from the ability to visualize and isolate cells at various stages of apoptosis. Schellenberger et al.(32)(33) and van Tilborg et al.(34) synthesized a series of functionalized magnetic-fluorescent nanoparticle conjugates that recognize apoptotic cells. The fluorophores involved in these bioconjugates, however, were not QDs but fluorescein isothiocyanate (FITC), which might limit the applications for these bioconjugates because of the disadvantages of this organic dye. A hallmark of apoptosis is the externalization of phosphatidylserine, which is recognized by annexin V. Accordingly, van Tilborg et al.(35) and Prinzen et al.(36) used annexin V–conjugated QDs coated with a paramagnetic lipid to detect apoptotic cells with fluorescence and magnetic resonance imaging. We recently demonstrated that trifunctional avidin-coupled nanospheres are capable of recognizing apoptotic cells preincubated with annexin V–biotin via the strong biotin-avidin interaction(31). To investigate the suitability of such trifunctional nanospheres for use in biomedical research, drug development, and/or diagnosis, we have conducted a detailed characterization of the properties of these trifunctional avidin-coupled nanospheres and applied them to visualizing and isolating apoptotic cells from a mixed cell population.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
reagents
The TACS Annexin V-Biotin Apoptosis Detection Kit was purchased from R&D Systems. Avidin, FITC-labeled biotin (FITC-biotin), and FITC-conjugated streptavidin (FITC-streptavidin) were obtained from Sigma-Aldrich. Coomassie Brilliant Blue G250 and BSA were purchased from Shanghai Bio Life Science & Technology. Ultrapure water (18 M{Omega}·cm) was produced with a WaterPro Plus water-purification system (Labconco).

preparation of fluorescent-magnetic-biotargeting trifunctional nanospheres
Monodisperse CdSe QDs were first obtained according to a published procedure(37). The core/shell CdSe/ZnS QDs(38) were then prepared by covering the core CdSe nanocrystals with a thin but higher band-gap material, ZnS, to yield QDs with strong light emission and high photostability. The nano-{gamma}-Fe2O3 particles and hydrazine-treated poly(styrene/acrylamide) nanospheres were prepared as previously described(30). FMBNs were subsequently prepared by swelling a 2-mL suspension of the hydrazine-treated poly(styrene/acrylamide) copolymer nanospheres, CdSe/ZnS QDs (3.0 mg), and nano-{gamma}-Fe2O3 particles (2.0 mg) in a chloroform/butanol solvent (5:95 by volume). The mixture was then ultrasonicated for 60 min, centrifuged for 5 min at 2790g, and washed 3 times with butanol to produce the poly(styrene/acrylamide) copolymer CdSe/ZnS-{gamma}-Fe2O3 bifunctional nanospheres.

We oxidized 0.4 mL of an avidin solution (5 g/L) with periodic acid according to a previously published method to create active aldehydes(39). The concentration of aldehyde-containing avidin was approximately 2.8 g/L according to the Bradford assay(40) measured with a TU1900 ultraviolet (UV)/visible spectrophotometer (Beijing Purkinje General Instrument). The aldehyde-containing avidin (240 µL) was then mixed with the FMBNs (2.4 mL of a 20.0-g/L suspension), diluted to 3.0 mL, and incubated for 6 h at room temperature. The nanospheres were washed 5 times with sodium phosphate buffer (0.1 mol/L, pH 6.8), and the resulting fluorescent-magnetic-biotargeting trifunctional avidin-coupled nanospheres were stored at 4 °C in sodium phosphate buffer (0.1 mol/L, pH 6.8).

measurement of fluorescence spectra
We measured the fluorescence spectra of QDs in n-hexane and the FMBNs in ultrapure water by means of a PerkinElmer LS 55 fluorescence spectrometer with a 388-nm laser for excitation and analyzed the results with FL WinLab 4.00.02 software (PerkinElmer).

measurement of the zeta potential of multifunctional nanospheres
The zeta potential is a measure of the potential at the interface between a solid surface and the liquid medium. We used a Zetasizer Nano ZS90 (Malvern Instruments) to measure the zeta potential of FMBNs before and after coupling avidin to their surfaces in 1x PBS (137 mmol/L NaCl, 2.7 mmol/L KCl, 10 mmol/L Na2HPO4, 1.8 mmol/LKH2PO4, pH 7.4).

measurement of protein on the surfaces of the trifunctional nanospheres
Aldehyde-containing avidin (20 µL of a 2.8-g/L solution) was mixed with 50 µL, 100 µL, 150 µL, or 200 µL of a 20.0-g/L suspension of FMBNs, diluted to 500 µL with sodium phosphate buffer (0.1 mol/L, pH 6.8), and incubated for 6 h at room temperature. We then centrifuged the suspensions, measured the amount of unbound avidin in the supernatants, and washed the nanospheres with sodium phosphate buffer (0.1 mol/L, pH 6.8) until the Bradford assay detected no residual protein in the supernatants(40). We then estimated the amount of nanosphere-bound avidin by subtracting the amount of unbound avidin from that in the starting solution.

cell culture
HeLa cells obtained from the China Center of Type Culture Collection were grown in DMEM with 100 mL/L fetal bovine serum. HeLa cells were irradiated with a 20-W UV lamp for 10 min to induce apoptosis, which was verified by flow cytometric analysis on an XL-MCL flow cytometer (Beckman Coulter) after the cells were stained first with propidium iodide (PI) and annexin V-biotin and then incubated with FITC-streptavidin in a 1x binding buffer (containing 1.8 mmol/L CaCl2, 10 mmol/L HEPES, 150 mmol/L NaCl, 5 mmol/L KCl, 1 mmol/L MgCl2, pH 7.4) according to the following protocol: washing cells with cold 1x PBS 3 times, suspending the cells in 100 µL incubation buffer [containing 1.0 mg/L annexin V-biotin and 10 µL of a concentrated solution (10x) of the binding buffer mentioned above] and incubating with 0.5 µg PI in the dark for 15 min at room temperature, suspending cells in the 100 µL binding buffer containing 20 µg FITC-streptavidin and then incubating in the dark for 15 min at room temperature, washing cells once with 100 µL 1x binding buffer, suspending them in 300 µL binding buffer and then analyzing the cells by flow cytometry (from the protocols provided by the manufacturer).

isolation of apoptotic hela cells with trifunctional avidin-coupled nanospheres
UV-irradiated HeLa cells were suspended in 100 µL incubation buffer as mentioned above. The cells then were incubated for 15 min at room temperature, washed 3 times with cold 1x PBS, and incubated with the trifunctional avidin-coupled nanospheres (20.0 g/L in 200 µL binding buffer) for 15 min at room temperature. The apoptotic HeLa cells bound to the trifunctional avidin-coupled nanospheres were precipitated with a magnet and then imaged with the aid of a fluorescence microscope. To demonstrate the nanospheres’ selectivity, we carried out a control experiment as outlined above except that we did not treat the cells with UV light. An additional control consisted of incubating irradiated cells with avidin-free nanospheres.

measurement of apoptotic cell isolation efficiency with trifunctional avidin-coupled nanospheres
The negative control consisted of suspending HeLa cells (1 x 105 ) not treated with UV light in 100 µL incubation buffer, incubating the cells with 0.5 µg PI in the dark for 15 min at room temperature, incubating the cells with 20 µg FITC-streptavidin in the dark for another 15 min at room temperature, and then analyzing the cells by flow cytometry. UV-treated HeLa cells (5 x 105 ) were suspended in 500 µL incubation buffer containing 2.5 µg PI, incubated in the dark for 15 min at room temperature, and then divided into 5 equal aliquots. One aliquot of cells was resuspended in 100 µL 1x binding buffer containing 20 µg FITC-streptavidin, incubated for 15 min in the dark at room temperature, and washed 3 times with cold 1x PBS. The cells were then collected and analyzed by flow cytometry to estimate the fraction of apoptotic cells in the aliquot of irradiated cells. The other 4 aliquots of cells were mixed with 50 µL, 100 µL, 150 µL, or 200 µL of the trifunctional nanospheres (20.0 g/L) and incubated in 1x binding buffer for 15 min in the dark. The cells captured by the nanospheres were then isolated with a magnet for 2 min and imaged with the aid of a fluorescence microscope. The cells remaining in the solution were incubated with 20 µg FITC-streptavidin and then analyzed by flow cytometry as described above to estimate the fraction of apoptotic cells remaining in each of the samples. The efficiency of the trifunctional nanospheres to isolate apoptotic cells was calculated from the difference in the fraction of apoptotic cells in cell samples treated and not treated with the trifunctional nanospheres.

imaging by fluorescence microscopy
All fluorescence micrographs were taken with the aid of an Olympus IX70 inverted fluorescence microscope coupled with a U-MWB filter cube (450–480 nm/500 nm/ 515 nm). Samples were spread on glass slides, placed on the microscope, visualized with a 100x oil-immersion objective or a 40x common objective, and imaged with a Nikon COOLPIX 5400 digital camera.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
characterization of the multifunctional nanospheres
Transmission electron microscopy of the FMBNs (Fig. 1A ) revealed that the QDs and the nano-{gamma}-Fe2O3 nanoparticles were approximately 2 nm and approximately 8 nm in diameter, respectively, and were well distributed in the poly(styrene/acrylamide) copolymer nanospheres [approximately 200 nm in diameter(30)]. An analysis of the fluorescence spectrum showed that the photoluminescence properties of the QDs were essentially unchanged in the FMBNs (Fig. 1B ), although the maximal emission spectra were redshifted by approximately 3–5 nm, compared with the free QDs in n-hexane solution. This slight redshift in the fluorescence-emission spectra might be because the QD environments in n-hexane and in the copolymer are different. Although the nanospheres undergo aggregation with storage, they are monodisperse and can retain the fluorescence property (Fig. 1, C and D ). To make FMBNs cell specific, we fabricated trifunctional avidin-coupled nanospheres by conjugating avidin to the surfaces of FMBNs, as we have reported previously(31) (Fig. 2A ).


Figure 1
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  		Figure 1. Characterization of FMBNs.

(A), transmission electron microscopy image. Thin arrows and thick arrows indicate the QDs and magnetic nano-{gamma}-Fe2O3 particles, respectively, embedded inside the nanospheres. (B), normalized fluorescence spectra of free green CdSe (a) and red CdSe/ZnS (c) QDs suspended in n-hexane solution and CdSe (b) and CdSe/ZnS (d) QDs embedded in nanospheres suspended in ultrapure water. Fluorescence microscopy with a 100x oil-immersion objective of green CdSe (C) or red CdSe/ZnS (D) QDs embedded in FMBNs. A.U., arbitrary unit.


Figure 2
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  		Figure 2. Characterization of fluorescent-magnetic-biotargeting trifunctional avidin-coupled nanospheres.

(A), schematic diagram of the nanospheres. (B), zeta potential distribution of FMBNs before (green curve) and after (red curve) coupling of avidin to their surfaces. (C), histogram for the amount of avidin molecules coupled to nanosphere surfaces under different conditions: 20 µL of a 2.8-g/L avidin solution reacting with 50 µL (a), 100 µL (b), 150 µL (c), or 200 µL (d) of the nanosphere suspension; data are presented as the mean (SD). (D–F), fluorescence images of trifunctional avidin-coupled nanospheres after incubation with FITC-biotin: (D), green fluorescence from FITC-biotin binding with avidin. (E), red fluorescence from QDs embedded inside the nanospheres after FITC photobleaching. (F), control reaction consisting of avidin-free FMBNs incubated with FITC-biotin; only a red fluorescence from the nanospheres was observed. K cps, kilo counts per second.

To understand the status of avidin on the nanospheres (including the uniformity of coupling, the amount, and bioactivity), we 1st studied the uniformity of avidin coupling to nanospheres by measuring the zeta potential of the nanospheres. Avidin has an isoelectric point of 10.5 and therefore is positively charged at pH 7.4. Consequently, the zeta potential of the avidin-coated nanospheres will be shifted positively compared with uncoated nanospheres. The results in Fig. 2BUp show that the FMBNs had peak zeta potentials of –27.26 mV and –11.15 mV before and after avidin coupling to their surfaces, respectively. The sharp pattern in the zeta potential distribution before and after avidin coupling indicates that the nanospheres had similar numbers of avidin molecules. We measured the amount of avidin immobilized on the surfaces of the nanospheres with a simple and rapid method based on the difference between the protein content of the starting solution and the amount remaining after avidin binding to the nanospheres. The results from 3 independent experiments are shown in Fig. 2CUp . Approximately 0.037 mg of avidin was coupled per milligram of nanospheres under our experimental conditions, regardless of the quantity of nanospheres used. These results suggest that a fixed number of sites on the nanosphere surface were available for coupling, a finding that agrees with the sharp pattern in the zeta potential distribution, which shows uniform coupling of avidin on all nanospheres. We subsequently confirmed that the avidin on the nanospheres specifically interacted with FITC-biotin, as we previously reported(31) (Fig. 2Up , D–F).

multifunctional nanosphere recognition of apoptotic cells at different stages of the apoptotic process
Our preliminary studies had shown that the avidin-coupled trifunctional nanospheres could bind apoptotic cells(31) preincubated with annexin V-biotin via avidin binding to biotin and annexin V binding to phosphatidylserine molecules exposed on the surfaces of the apoptotic cells (Fig. 3 ). Cells in the early stages of apoptosis can be distinguished from late-apoptotic/necrotic cells by their ability to be labeled with annexin V but not with the DNA-binding organic fluorescent dye PI, whereas late-apoptotic cells can be labeled with both annexin V and PI. Thus, we investigated the potential for such trifunctional nanospheres to identify apoptotic cells at different stages of the apoptotic process and for efficiently isolating apoptotic cells from living cells. The cell in Fig. 4A has a morphology characteristic of cells in early apoptosis, an observation in agreement with the intense green fluorescence (from the trifunctional nanospheres) at the periphery of the cell. The cell also exhibited a weak red fluorescence (from PI) in the periphery of the nucleolus, because the lack of damage to the nuclear membrane prevented PI from entering the nucleus (Fig. 4B ). In contrast, the cell in Fig. 4D shows an intense green fluorescence from the trifunctional nanospheres and an intense red fluorescence from PI, indicating the presence of phosphatidylserine on the cell surface and the membrane damage associated with late-stage apoptosis (Fig. 4C ). It is evident that these multifunctional nanospheres in combination with PI staining of nuclear DNA can effectively distinguish apoptotic cells at different stages of the apoptotic process. This method may be helpful for understanding the progression of apoptosis and in analyzing apoptosis-associated changes in DNA, proteins, or organelles.


Figure 3
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  		Figure 3. Microscopy images of apoptotic HeLa cells captured by trifunctional avidin-coupled nanospheres.

(A), bright-field image of nanospheres bound to cell surfaces. (B), green fluorescence from QDs embedded inside the nanospheres bound to the cells. (C–F), control experiments: HeLa cells without UV irradiation incubated with trifunctional avidin-coupled nanospheres (C and D); apoptotic HeLa cells incubated with avidin-free FMBNs (E, F). No nanospheres (green fluorescence) were found on cells in these control experiments.


Figure 4
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  		Figure 4. HeLa cell fluorescence at different apoptotic stages for cells preincubated with annexin V-biotin and PI and then treated with avidin-containing trifunctional nanospheres and magnetic isolation.

(A and C), bright-field and (B and D) fluorescence images of the cells irradiated with UV light for 10 min and incubated for 12 h (A and B) or 48 h (C and D).

efficient isolation of target cells with multifunctional nanospheres
As we demonstrated previously(30)(31), the magnetic property of multifunctional nanospheres can be helpful for isolating specific cells. We used flow cytometry to obtain the fraction of apoptotic cells before and after isolation with multifunctional nanospheres to estimate the efficiency of the multifunctional nanospheres for separating apoptotic cells from living cells. Apoptosis-induced HeLa cells were treated as described in Materials and Methods and isolated with the aid of a magnet. The entire process, including cell recognition and magnetic isolation, could be accomplished within 35 min. The cells remaining in the solution were then analyzed by flow cytometry after incubation with FITC-streptavidin. The results showed that in the absence of UV irradiation, few cells were in the apoptotic region (Fig. 5A , upper-right quadrant), and the vast majority was in the region of live cells (Fig. 5A , lower-left quadrant). UV irradiation produced an appreciable and increasing proportion of the cells in the apoptotic region (Fig. 5B , upper-right quadrant), as reflected by the increases in the FITC signal (vertical axis) due to FITC-streptavidin binding to annexin V–biotin bound to the apoptotic cells and in the PI signal (horizontal axis) due to PI binding to nuclear DNA. Incubating the UV-irradiated cells with the nanospheres and precipitating the nanospheres with a magnet produced a cell-distribution pattern (Fig. 5C ) in the flow cytometry analysis similar to that produced by nonirradiated HeLa cells (Fig. 5A ), suggesting that nearly all apoptotic cells were removed at all of the nanosphere concentrations used (50–200 µL of a 20.0-g/L nanosphere suspension; Fig. 5D ). We carried out a quantitative analysis by using the equation:

Formula
where Es is the isolation efficiency and Fb and Fa are the fractions of apoptotic cells in the total sample before and after isolation with multifunctional nanospheres, respectively. The flow cytometric analysis revealed an isolation efficiency for apoptotic cells of approximately 80%; that is, the trifunctional nanospheres removed approximately 80% of the apoptotic cells at all nanosphere concentrations used, with the exception of the highest nanosphere concentration (Fig. 5E ). This last result was likely due to the characteristics of the nanospheres themselves. Our trifunctional avidin-coupled nanospheres were made from poly(styrene/acrylamide) infused with nano-{gamma}-Fe2O3 particles and QDs. The materials sometimes caused unwanted aggregation. Some factors (i.e., nanosphere size, surface charge level, and concentration) will produce aggregation. For very small nanospheres such as ours, maintaining a monodisperse suspension was relatively easy at low nanosphere concentrations without the addition of a surfactant, but as the nanosphere concentration in the suspension increases, the likelihood of collisions and hydrophobic interactions increases. Such interactions can lead to partial aggregation. Partial nanosphere aggregation can decrease the amount of the immobilized proteins accessible to binding by cells in the mixture and decrease Brownian motion, again leading to a reduced probability of the trifunctional nanospheres to bind to apoptotic cells. In any case, our results show that these multifunctional nanospheres can be used to isolate target cells from samples at an efficiency of approximately 80% or greater.


Figure 5
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  		Figure 5. Flow cytometric analysis of HeLa cells.

(A), negative control: typical nonirradiated HeLa cells not incubated with trifunctional nanospheres. (B), positive control: UV-irradiated HeLa cells not incubated with nanospheres. (C), UV-irradiated HeLa cells after isolating the nanosphere-bound apoptotic cells with a magnet. (D), histogram for the fraction of apoptotic cells before (solid columns) and after (open columns) isolation with 50 µL (a), 100 µL (b), 150 µL (c), or 200 µL (d) of multifunctional nanospheres; data are presented as the mean (SD) of 3 independent measurements. (E), histogram for the efficiency of isolation of UV-irradiated HeLa cells after incubation with 50 µL (a), 100 µL (b), 150 µL (c), or 200 µL (d) of trifunctional nanospheres, isolation with a magnet, and flow cytometric analysis; data are presented as the mean (SD) of 3 independent measurements.

In conclusion, our previous studies demonstrated that the fluorescent-magnetic multifunctional avidin-coupled nanospheres could bind to apoptotic cells prebound to annexin V-biotin. In the present detailed characterization of these nanospheres, we have demonstrated that these nanospheres retained the fluorescence spectra of the individual QDs. We also have demonstrated the easily monodisperse nature of the nanospheres and thus their suitability for biomedical applications that typically require a uniform distribution of nanomaterials. We also have shown that a fixed amount of avidin was coupled to per milligram nanospheres. Such characteristics are particularly important in many biomedical applications, especially when quantification is required. Finally and more importantly, we have shown in an example of a potential use for our nanospheres that the combination of annexin V-biotin and PI with the trifunctional nanospheres enables the visualization of apoptotic cells at different stages of apoptosis and that we can isolate apoptotic cells from living cells with an efficiency of at least 80%. Obviously, such nanospheres have superior properties of fluorescence, magnetism, specificity, monodispersity, and agility in colliding and interacting with large species. We envision that the choice of biotargeting molecules other than avidin and/or the use of different biotin-coupled molecules may make trifunctional nanospheres useful for visually identifying and isolating other target cells at various stages of development, such as tumor cells, activated macrophages, and even bacteria and viruses, for purposes of biochemical, molecular, and genetic analyses, clinical diagnosis, drug development, and so on. In addition, multifunctional nanospheres may become useful for synchronous diagnosis of multiple diseases, the identification and collection of multiple bacteria or viruses, and simultaneous magnetic resonance and fluorescence imaging owing to the nanospheres’ fluorescence (e.g., multicolor labeling and single-excitation/multiple-emission) and magnetic traits.


   Acknowledgments
 
Grant/funding support: This work was supported by the 863 Program (no. 2006AA03Z320), the National Key Scientific Programs—Nanoscience and Nanotechnology (no. 2006CB933100), the Science Fund for Creative Research Groups (no. 20621502), the National Natural Science Foundation of China (Grant nos. 20505001 and 30570490), the Ministry of Education (nos. 306011 and IRT0543), and the Beijing Institute of Technology Fund for Excellent Youth (000Y06-24). This research was also supported in part by the Intramural Research Program of the National Institute of Child Health and Human Development, National Institutes of Health.

Financial disclosures: None declared.

Acknowledgments: We thank Wei Dong and Ming-Xi Zhang for their kind help.


   Footnotes
 
1 Nonstandard abbreviations: QD, quantum dot; FMBN, fluorescent-magnetic bifunctional nanosphere; FITC, fluorescein isothiocyanate; FITC-biotin, FITC-labeled biotin; FITC-conjugated streptavidin; UV, ultraviolet; PI, propidium iodide.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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E.-Q. Song, Z.-L. Zhang, Q.-Y. Luo, W. Lu, Y.-B. Shi, and D.-W. Pang
Tumor Cell Targeting Using Folate-Conjugated Fluorescent Quantum Dots and Receptor-Mediated Endocytosis
Clin. Chem., May 1, 2009; 55(5): 955 - 963.
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