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A Mild and Reliable Method to Label Enveloped Virus with Quantum Dots by Copper-Free Click Chemistry Jian Hao,†,§ Li-Li Huang,†,§ Rui Zhang,† Han-Zhong Wang,‡ and Hai-Yan Xie*,† †

School of Life Science, Beijing Institute of Technology, Beijing 100081, China Wuhan Institute of Virology, Chinese Academy of Sciences, Wu Han 430071, China

‡

S Supporting Information *

ABSTRACT: Real-time tracking of the dynamic process of virus invasion is crucial to understanding the infection mechanism. For successful tracking, efficient labeling methods are indispensable. In this paper, we report a mild and reliable method for labeling viruses, especially with regard to easily disabled enveloped viruses. The copper-free click chemistry has been used to label enveloped viruses with quantum dots (QDs) by linking virions modified with azide to the QDs derived with dibenzocyclooctynes (DBCO). Both vaccinia virus (VACV) and avian influenza A virus (H9N2) can be specifically and rapidly labeled under mild conditions, with a labeling efficiency of more than 80%. The labeled virions were of intact infectivity, and their fluorescence was strong enough to realize single-virion tracking. Compared to previously reported methods, our method is less destructive, reliable, and universal, without specific requirements for the type and structure of viruses to be labeled, which has laid the foundation for long-term dynamic visualization of virus infection process.

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multicolor labeling. Anyway, viruses usually need to be fluorescently labeled first. Generally speaking, a perfect virus labeling method must be mild and reliable enough to ensure the virus is infectious after labeling; that is, labeling will not affect the virus invasion. At the same time, the label signal should be strong and photostable enough for long-term tracking. But there are shortcomings with the existing methods based on both green fluorescent protein (GFP) fusion labeling10−12 and organic fluorescent dye conjugation.13,14 Therefore, it is urgent to develop reliable virus labeling methods, while quantum dots (QDs) provide us an opportunity to solve such a labeling problem because of their high fluorescence brightness and photostability.15−17 Existing methods for labeling enveloped viruses with QDs as a fluorophor are substantially based on the biotin−avidin affinity system, which is highly efficient and specific. But conjugating the QDs with streptavidin will remarkably increase the size of QDs, which might block the recognition site and change the movement behaviors of viruses to some extent, especially for small-sized viruses. Click chemistry ligations take place between small chemical groups, not to notably increase the size of QDs. Besides, they are rapid, highly selective and efficient, and occur under physiological conditions. Therefore, they are of high importance in bioconjugate chemistry field. Up

nderstanding the mechanism of virus invasion is crucial to prevention and control of viral diseases, which has not been fully understood because of the diversity of viruses and complexity of their invasion processes. Generally, it is difficult to draw definite conclusions about the mechanisms of virus infection due to the limitation of conventional in vitro nondynamic methods commonly used 10 years ago. For example, early studies using electron microscopy and cell fractionation technology suggested that poliovirus entered cells via clathrin-mediated endocytosis1,2 and human immunodeficiency virus (HIV) required fusion between viral and cellular membranes to insert viral genome into host cells.3,4 However, Zhuang and co-workers5 found that poliovirus entered the cells by a clathrin-independent but tyrosine kinase- and actindependent endocytosis mechanism by real-time living cell fluorescence imaging. Using similar technology, Melikyan and co-workers6 also found that HIV entered cells via endocytosis, after which the endocytosis bubbles fused with endosomes by dynamin-dependent mechanism. Obviously, real-time tracking of virus infection process based on the emerging dynamic imaging technology has provided opportunities to accurately elucidate virus infection mechanism. Since the movement speed of viruses is not so fast, normally with a speed on the order of micrometers per second,7−9 the existing high-speed microscopy imaging technology with an image-acquisition speed of up to 2000 frames per second makes it possible to track the dynamic biological process of viruses in four dimensions, or even in five dimensions based on © 2012 American Chemical Society

Received: July 9, 2012 Accepted: September 4, 2012 Published: September 4, 2012 8364

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to date, only a few reports on labeling of viruses18−20 by click chemistry appeared, based just on copper-catalyzed ligation. However, in the case of copper-catalyzed ligation the Cu(II)− triazoles adduct, produced by the interaction between Cu2+ and 1,2,3-triazoles, might unfavorably decompose the structure of viruses.18 On the other hand, Cu2+ can greatly decrease the fluorescence of the QDs.21,22 Evidently, it is not feasible to label viruses with QDs just by copper-catalyzed ligation. But it is known that many animal viruses are enveloped. Actually, there are few reports on labeling of enveloped viruses by click chemistry, most probably because the structure of enveloped viruses is much more fragile than that of nonenveloped viruses and cannot endure many chemical reactions. Overall, it has still been a problem to label enveloped viruses. Accordingly, alternative approaches to overcome potential Cu(I/II) toxicity, such as copper-free click chemistry, are being developed. Copper-free click chemistry (also termed strain-promoted alkyne−azide cycloaddition, SPAAC) is very promising in biolabeling because of its nontoxicity and biocompatibility. Bertozzi and co-workers23−26 labeled cells, zebrafish, and mice by copper-free click chemistry with difluorinated cyclooctyne (DIFO) and its derivatives and studied the dynamics of glycan trafficking. Boons and co-workers27,28 and van Delft and coworkers29,30 greatly promoted copper-free click chemistry by developing several cyclooctyne derivatives, such as 4dibenzocyclooctynol (DIBO, DBCO), azadibenzocyclooctyne (ADIBO), and bicyclo[6.1.0]nonyne (BCN), which further increased the reactivity. Recently, Zhang et al.31 reported the feasibility of labeling baculovirus with QDs by copper-free click chemistry. However, much work still needs to be done to establish reliable methods to mildly label enveloped viruses. In this paper, copper-free click chemistry has been used to label enveloped viruses with QDs by linking virions surfacemodified with azide to the QDs derived with dibenzocyclooctynes (DBCO). The viruses can be specifically and rapidly labeled with QDs under mild conditions. The labeling efficiency is more than 80%. The infectivity of viruses was scarcely affected. The fluorescence of the labeled virions was strong enough to facilitate tracking of single virions. Compared to biotin−streptavidin affinity-based labeling or the coppercatalyzed ligation method, our method has superiorities, including no increase in QD size, stable fluorescence of QDs, and intact infectivity of viruses. Therefore, our method is reliable and universal, without specific requirements for the type and structure of viruses to be labeled.

605 nm) were purchased from Invitrogen (Carlsbad, CA). All other chemical reagents were supplied by Beijing Chemical Reagent Co. Preparation and Characterization of QDS-DBCO. Five microliters of 8 μM QDS-NH2 was mixed with 5 μL of 36 mM DBCO-PEG4-NHS ester in 40 μL of phosphate-buffered saline (PBS; pH 7.2, 0.01 M) for about 3 h at room temperature. Excessive DBCO-PEG4-NHS ester was removed with a desalting NAP-5 column (GE Healthcare). The obtained QDS-DBCO was identified by gel electrophoresis (DYY-6C, China) in 1% agarose gel with 0.5× Tris−borate−ethylenediaminetetraacetic acid (EDTA) buffer (TBE) as the electrophoretic buffer. Spectroscopic properties of QDSDBCO and QDS-NH2 were characterized with a ultraviolet− visible spectrophotometer (U-3900, Hitach, Japan) and a fluorescence spectroscope (FluoroMax-4) with an excitation wavelength of 450 nm. The hydrodynamic particle sizes of the QDs were measured by dynamic light scattering (DLS) (Zetasizer Nano ZS90, Malvern Instruments). Preparation of Azide-Coated or DBCO-Coated Glass Slides. Glass slides (22 mm × 22 mm, Citoglas) were immersed in a solution of poly-L-lysine (Sigma) overnight at 37 °C. The slides were then rinsed three times with PBS and subsequently immersed in a solution of 100 μM azide-PEG4NHS ester or DBCO-PEG4-NHS ester for 2 h at 37 °C. Cell Culture and Virus Propagation. Vero cells were maintained in media according to the instructions of the American Type Culture Collection. VACV was propagated in monolayer cultures of Vero cells at 37 °C in the presence of 2% fetal bovine serum (FBS). The cytopathic effect (CPE) of VACV was assessed 1−2 days postinfection prior to removal of cell debris by centrifugation (2000g for 15 min) at 4 °C. VACV was purified by differential centrifugation and density gradient centrifugation as previously described.16 H9N2 was propagated in the allantoic cavity of 10-day-old embryonated eggs for 48 h at 37 °C. Subsequently, H9N2 was purified as previously described.17 Virus Titer Assays. The titer of VACV was quantified by 50% tissue culture infective dose (TCID50). Vero cells were cultured in 96-well plates in culture medium until the cells reached 80−90% confluence. The virus samples and their paired controls were immediately serially diluted with Hanksʼ balanced salt solution supplemented with 25 mM N-(2hydroxyethyl)piperazine-N′-ethanesulfonic acid (Hepes) buffer and 4 mM sodium bicarbonate at pH 7.2. They were subsequently added to the cells and infected for 1 h. TCID50 cultures were washed and fed with Dulbeccoʼs modified Eagleʼs medium (DMEM) containing 2% FBS. Then the infected cells were cultured in an incubator at 37 °C with CO2 for about 5 days. The number of wells that have CPE on Vero cells were counted. TCID50 was calculated from the Reed−Muench formula: TCID50 = dilution above 50% CPE + [(% next above 50%) − 50%]/(% next above 50% − (% next below 50%) × log 10. Preparation of QD-Labeled Virus. To label the viruses with QDs, the viruses were modified first with azide or biotin in PBS (pH 7.2, 0.01 M). The viruses (about 1 × 107 TCID50/ mL) were incubated with azide-PEG4-NHS ester (100 μM) or EZ-Link sulfo-NHS-LC-biotin (1 mg/mL) for 1 h at room temperature. Unreacted azide-PEG4-NHS ester or EZ-Link sulfo-NHS-LC-biotin was removed with a desalting NAP-5 column. Subsequently, the modified viruses were labeled with

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EXPERIMENTAL SECTION Materials and Reagents. Vaccinia virus (VACV) tiantan strain and avian influenza A virus (H9N2) strain were obtained from Wuhan Institute of Virology, Chinese Academy of Sciences. Vero cells (African green monkey kidney cells) were purchased from Peking Union Medical College Hospital; azide-PEG4-NHS ester and DBCO-PEG4-NHS ester from Click Chemistry Tools (Scottsdale, AZ); Dylight 488 conjugated goat anti-mouse IgG antibody from Earthox LLC (San Francisco, CA); anti-H3 protein (vaccinia virus) mouse monoclonal antibody (mAb) from Immune Technology Corp.; antihemagglutinin (anti-HA) protein (H9N2) mAb from Sino Biological Inc. (Beijing, China); and EZ-Link sulfo-NHS-LCbiotin from Pierce Biotechnology. Qdot 605 ITK amino [poly(ethylene glycol), PEG] quantum dots (QDS-NH2, 605 nm), Qdot 605 streptavidin (SA) conjugate (QDS-SA, 605 nm), and Qdot 605 ITK carboxyl quantum dots (QDS-COOH, 8365

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RESULTS AND DISCUSSION Modification of the QDs with DBCO. As shown in Figure 1, QDs should be modified first with dibenzocyclooctynes

QDs by incubation with 10 nM QDS-DBCO or QDS-SA for 1 h at room temperature. Virus Growth Curves. Vero cells in a monolayer were infected with VACV, QD-biotin-VACV, and QD-azide-VACV at multiplicity of infection (MOI) = 5 for 4, 8, 12, 16, 20, and 24 h. The infected cells were scraped into medium, after three rounds of freeze−thaw, and the cell debris was removed by centrifugation at 2000g for 15 min. The infectivities of collected virions were determined by TCID50. Flow Cytometry Analysis of Viral Infectivity. Vero cells were seeded in 6-well culture dishes (2.6 × 105 cells/well) and infected with as-prepared virus for 12 h before flow cytometry (BD Calibur) assay of GFP+ cells. All samples were counted over 10 000 cells, and all data were processed with WinMDI and Origin 8.0. Fluorescence Colocalization Assay of Virus on Glass Slides. The purified wild-type virus or azide-modified virus solution was dropped onto slides (22 mm × 22 mm, Citoglas) coated by anti-H3 protein (VACV) mouse mAb or anti-HA protein (H9N2) mouse mAb. Then the virus was fixed with heat. After being blocked with 2% (w/v) bovine serum albumin (BSA), the virus was labeled with QDS-DBCO. The nucleic acid of the virus was stained with Hoechst 33342 or SYTO 82 by incubation with 2 mg/mL Hoechst 33342 or 10 mM SYTO 82 solutions for 30 min. Excess Hoechst 33342, SYTO 82, and QDS-DBCO were washed out by 1× PBS. The fluorescence images were acquired on a Leica laser confocal fluorescence microscope (TCS SP5). In VACV labeling, the QDs were excited by use of a 488 nm laser, emitting 600−650 nm fluorescence. Hoechst 33342 was excited with UV, emitting 450−500 nm fluorescence. In H9N2 labeling, the QDs were excited with UV, emitting 600−650 nm fluorescence. SYTO 82 was excited with a 543 nm laser, emitting 550−570 nm fluorescence. Immunofluorescence Assay of Virus. Cells grown to 50% confluence were incubated in 35-mm glass-bottom culture dishes (NEST Corp.) with as-prepared QD-labeled virus for 30 min at 4 °C to allow virus binding. The excessive QD-virus was washed out by 1× PBS. Then the cells were fixed with 4% paraformaldehyde for 15 min at room temperature. After being blocked in PBST containing 1% BSA for 1 h at room temperature, the samples were incubated with anti-H3 protein (VACV) or anti-HA protein (H9N2) mouse mAb for 1 h at room temperature, followed by incubation with Dylight 488 conjugated goat anti-mouse IgG antibody for 1 h and then with 10 μg/mL Hoechst 33342 for 10 min at room temperature in the dark. The samples were rinsed 3−4 times with PBS between two successive steps. The fluorescence images were acquired on a Leica laser confocal fluorescence microscope (TCS SP5). Dylight 488 was excited with a 488 nm laser, and 500−530 nm wavelength was used for emission. Dynamic Tracking of Viruses. The dynamic behaviors of the viruses were fluorescently tracked with a spinning-disk confocal microscope (Andor Revolution XD), equipped with an Olympus IX 81 microscope, a Nipkow disk-type confocal unit (CSU 22, Yokogawa), a CO2 online culture system (INUBG2-PI), and an electron microscope charge-coupled device (EMCCD) (Andor iXon DV885K single photon detector). The QDs605 were detected using continuous excitation with 488 nm laser, and their signals were collected through a 605/35 nm band-pass filter.

Figure 1. Schematic illustration of the labeling strategy.

(DBCO) to obtain the functionalized QDs (QDS-DBCO) for the following copper-free click chemistry. Amino (PEG) QDs (QDS-NH2) were allowed to react with DBCO-PEG4-NHS ester, whose carboxyl group had been activated with Nhydroxysuccinimide (NHS). The reactive ester can react with amino rapidly under mild conditions. The formation of QDSDBCO can be confirmed by electrophoresis because the QDs will decrease in positive charges due to depletion of amino groups on their surface and increase in weight after the modification. As shown in Figure 2a, QDS-DBCO ran obviously faster than QDs, indicating that the effect of the positive charge decrease surpassed that of the weight increase. The spectra hardly changed after the modification (Figure 2b). DBCO can specifically react with azide by the ring-strain-promoted cycloaddition of alkynes with azide. When QDS-DBCO was

Figure 2. (a) Agarose gel electrophoresis of QDs: (lane 1) QDS-NH2 and (lane 2) QDS-DBCO. (b) Spectra of QDs. (c) Confocal microscopic fluorescence imaging of glass slides. The glass slides were first coated with azide or DBCO, then QDS-DBCO or QDS-NH2 was added and kept for 1 h. The unreacted QDS-DBCO or QDS-NH2 was washed with PBS. (d) Hydrodynamic diameter profiles of QDs. 8366

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dropped onto glass slides covered with azide or DBCO, they could only be immobilized on the surface of glass slides covered with azide. QDS-NH2 could not immobilize on the surface of glass slides covered with azide (Figure 2c). So it can be concluded that the DBCO-QDs had been successfully prepared because they showed both the unique fluorescence property of QDs and the reaction specificity of DBCO with azide. In general, the labeling for dynamic tracking of virus invasion process should influence neither the recognition ability nor the infectivity of the virus for the host cells. Therefore, the smaller the nanoparticles used as a tag are, the slighter the unfavorable influence is. A popular method based on an affinity system was realized by interaction between streptavidin conjugated to QDs (QDS-SA) and biotin bound to virus (biotin-virus). However, the size of QDs notably increased after their conjugation with streptavidin, typically from 16 to 24 nm, because of the large size of streptavidin (Figure 2d). Fortunately, there was no size increase of the QDs when they were conjugated with DBCO. On the contrary, the size of QDS-DBCO (13.6 nm) decreased by 15% compared to that of the QDs (16 nm) (Figure 2d), which is consistent with the reported result.21 This may be because the DBCO is hydrophobic, decreasing the quantity of hydrated water molecules on the surface of QDS-DBCO. The size decrease will lessen the steric hindrance to the recognition site and the distortion of virus infection behaviors as much as possible. Modification of Virus with Azide. To label the virus with QDs through copper-free click chemistry, the virus needs to be modified first with azide. Similar to the modification of QDs, the azide-PEG4-NHS ester was used to react with amino groups on the virus surface to produce azide-modified virus (azidevirus). At first, the recombinant vaccinia virus (VACV) carrying a green fluorescent protein (GFP) reporter gene was used in our experiments.32,33 Vero cells exhibited GFP expression after having been infected by this virus, whose infectivity can be accordingly evaluated. As can be seen in Figure 3a,b, when the concentration of the azide-PEG4-NHS ester was lower than 100 μM, the infectivity of the virus remained more than 90% that of the control. The infectivity of the virus would decrease to a certain extent when the concentration of azide-PEG4-NHS ester continued to increase. Therefore, the concentration was fixed at 100 μM to modify the virus with azide. To study the infectivity of azide-virus, the Vero cells were infected by this virus. As can be seen from Figure 3c, from 12 to 48 h, the quantities of GFP expression had no obvious difference between the modified VACV and the control. The titers of wild-type VACV and azide-VACV of the same batch were 1.74 × 107 and 1.45 × 107 TCID50/mL, respectively, which had no significant difference. The titer of the biotinVACV of the same batch was 1.15 × 107 TCID50/mL, which is lower than that of the azide-VACV (Figure 4a). In the one-step growth curve (Figure 4b), azide-VACV had no significant decrease in growth compared to wild-type VACV. So it could be concluded that the azide modification had little influence on the infectivity of the virus. The modified virus was still fully infectious. Since the azide modification makes use of the amino groups that are ubiquitous on the surface of all kinds of viruses, this modification method is universal for all viruses. Labeling Azide-Virus with QDS-DBCO by Copper-Free Click Chemistry. The labeling could be easily realized just by mixing azide-virus with QDS-DBCO. It is known that azide and DBCO can react with each other rapidly and efficiently under mild conditions, producing the QD-labeled VACV (QD-

Figure 3. (a) Flow cytometry plots of Vero cells infected by azideVACV. Different VACV samples of the same batch were respectively incubated with azide-PEG4-NHS ester at different concentrations for 1 h to realize the modification. Subsequently, the Vero cells were infected by azide-VACV for 12 h before flow cytometry assay of GFP+ cells. All samples were counted over 10 000 cells, and all data were processed with WinMDI and Origin 8.0. (b) Percentage of GFP+ cells calculated from panel a. Data from three independent experiments were normalized as a percentage of the control. *p = 0.05; **p = 0.01. (c) Confocal microscopic fluorescence imaging of Vero cells infected by azide-VACV at MOI = 10 for various times (panels a′−d′ and a1′− d1′, azide-VACV; panels e′−h′ and e1′−h1′, wild-type VACV; Cazide‑PEG4‑NHS ester = 100 μM; panels a′−h′, bright-field; panels a1′− h1′, fluorescence of GFP).

Figure 4. (a) Titers of viruses. Vero cells were cultured until they reached 80−90% confluence. Serially diluted virus samples and their corresponding controls were added to the cells and infected for 1 h postinfection. TCID50 cultures were washed and fed with DMEM containing 2% FBS. Then the infected cells were cultured in an incubator at 37 °C with CO2 for about 5 days. The number of wells that have CPE on Vero cells was counted. TCID50 in Vero cells was calculated from the Reed−Muench formula: TCID50 = dilution above 50% CPE + [(% next above 50%) − 50%]/(% next above 50% − (% next below 50%) × log 10. (b) Growth curves of viruses. Vero cells were infected at MOI = 5 at the indicated times postinfection. Cells were frozen and thawed three times, sonicated, and centrifugated. Titers were determined by TCID50.

VACV). The labeled virions were so strong in fluorescence that single-virion imaging could be easily realized. The labeling efficiency was evaluated by fluorescence microscopic colocalization assay of the virions, whose DNA was labeled with Hoechst 33342. As can be seen from Figure 5a−c, most fluorescence signals of Hoechst 33342 were colocalized with those of QDs. The colocalization efficiency of the resulted virions was (88 ± 2)% (mean ± SD). On the contrary, no QD signal was observed for the virions unmodified with azide (Figure 5a1−c1). Therefore, this labeling method was not only highly efficient but also highly specific. 8367

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with Vero cells. The fluorescence microscopic colocalization imaging clearly showed that most fluorescence signals of the QDs had been colocalized with that of Dylight 488 bound to the H3 protein, one of the envelope proteins of VACV. The colocalization efficiency of the labeled virions was (87 ± 6)% (mean ± SD). On the contrary, there was no signal of the QDs when the cells were coincubated with a mixture of wild-type VACV and QDs (Figure 7), suggesting that there was no

Figure 5. Fluorescence colocalization of the signals of QDs linked to VACV and the Hoechst 33342 intercalating the nucleic acid of the VACV. Wild-type VACV or azide-VACV solution was dropped onto slides coated with anti-H3 protein (VACV) mouse mAb. Then the VACV was fixed with heat. After being blocked with 2% (w/v) BSA, the VACV was labeled with QDS-DBCO. The nucleic acid of the VACV was stained with Hoechst 33342. Excess Hoechst 33342 and QDS-DBCO were washed out by 1× PBS. The QDs were excited by use of a 488 nm laser, emitting 600−650 nm fluorescence. Hoechst 33342 was excited with UV, emitting 450−500 nm fluorescence. (a−c, azide-VACV; a1−c1, wild-type VACV; green, Hoechst 33342; red, QDs; yellow, merge).

Figure 7. Fluorescence imaging of cells coincubated with QD-VACV (a−d) or a mixture of VACV and QDS-DBCO (a1−d1). The H3 protein, one of the envelope proteins of VACV, had been labeled with Dylight 488 through indirect immunofluorescence labeling strategy. The nucleus of the cells was stained with Hoechst 33342. (Blue, Hoechst 33342; green, Dylight 488; red, QDs.)

Quantitative spectroscopic analysis results showed that the fluorescence intensity of the QDs was retained by about 85% after they had reacted with azide-VACV for 1 h (Figure 6a),

obvious nonspecific adsorption of the QDs on the surface of the cells. Evidently, the interaction between QD-VACV and Vero cells should be attributed to preservation of the specific recognition ability of the labeled virions. Actually, whether the labeled virus is infectious or not is the most important problem in the present work. The infectivity of QD-VACV can be evaluated by virus titer assay and flow cytometry (Figure 8). Titers of QD-VACV and wild-type VACV of the same batch were 7.36 × 106 and 1.74 × 107

Figure 6. (a) Photoluminescence (PL) intensity of 10 nM QDSDBCO coincubated with azide-VACV or wild-type VACV for 1 h. (b) PL spectra of 10 nM QDS-DBCO reacted with azide-VACV for 1 min under different conditions.

indicating that the copper-free click chemistry less influenced the fluorescence of QDs. In the case of the typical coppercatalyzed ligation, both Cu2+ and ascorbate are used at concentrations of a few micromolar. Ascorbate reduces Cu2+ to Cu+, and subsequently, Cu+ catalyzes the cycloaddition reaction between azide and alkyne. As can be seen from Figure 6a, even if only 1 μM Cu2+ was present, the fluorescence intensity of the QDs decreased by more than 80% after QDSDBCO had been mixed with azide-VACV for only 1 min. And the fluorescence was hardly detectable if the time lasted for 10 min. It is obvious that, in the case of the typical coppercatalyzed ligation, the fluorescence of the QDs will be heavily decreased. Microscopic imaging of the cells coincubated with asprepared QD-VACV showed remarkable signals of the QDs on the cytomembrane, indicating that QD-VACV could interact

Figure 8. (a) Titers of VACV. (b) Flow cytometry plots of Vero cells infected by VACV. Different VACV samples under different conditions as stated were used to infect the Vero cells for 12 h before flow cytometry assay of GFP+ cells. All samples were counted over 10 000 cells, and all data were processed with WinMDI and Origin 8.0. (c) Percentage of GFP+ cells calculated from panel b. Data from three independent experiments were normalized as a percentage of the control. **p = 0.01. 8368

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Author Contributions

TCID50/mL, respectively, which were of no significant difference. For the virus QD-labeled by biotin−streptavidin affinity (QD-biotin-VACV), the titer was 4.31 × 106 TCID50/ mL, which was lower than that of the virus QD-labeled by copper-free click chemistry (QD-azide-VACV). On the basis of GFP expression detected by flow cytometry, it was found that the bioactivity of the virus decreased by about 50% in the presence of Cu2+ and decreased by 82% in the presence of both Cu2+ and ascorbate. All the results adequately indicated that the infectivity of QD-VACV prepared by copper-free click chemistry could be maintained. Hence, the copper-free click chemistry-based method is preferable for labeling enveloped viruses with QDs, because it is milder and more reliable than the affinity system-based and copper-catalyzed ligation methods. Again, these data validated that copper-free click chemistry could be efficiently applied to labeling of enveloped viruses. Since QD-VACV was of both strong fluorescence and intact infectivity, the dynamic behavior of the virus can be conveniently tracked by use of a living-cell high-speed microscopic imaging system (see Movie S-1 in Supporting Information). Because all kinds of viruses have lots of amino groups on their surface, they all can be modified with the azide-PEG4NHS ester. Therefore, this labeling method can be generalized to label diverse viruses. Similar results were obtained when another virus, H9N2 influenza virus, was labeled by this method. For the labeled virions, the fluorescence microscopic colocalization efficiency between SYTO 82 bound to RNA and QDs linked to the virus was (86 ± 6)% (mean ± SD) (see Figure S-1 in Supporting Information). Further, most fluorescence signals of Dylight 488 bound to the HA protein, one of the envelope proteins of H9N2, could be colocalized with that of QDs linked to the virus. The colocalization efficiency of the labeled virions was (88 ± 2)% (mean ± SD) (see Figure S-2 in Supporting Information). The dynamic behaviors of single virions can also be tracked (see Movie S-2 in Supporting Information).

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J.H. and L.-L.H. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Basic Research Program of China (973 Program, 2011CB933600), the National Natural Science Foundation of China (20975013), and the Program for New Century Excellent Talents in University (NCET-08-0046). We thank Bi-Hai Huang and ShuLin Liu for their helpful discussion.

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CONCLUSIONS In this paper, a QD-based labeling method for enveloped viruses through copper-free click chemistry has been studied in detail. Viruses can be labeled, with high efficiency and specificity, by first modifying with azide and subsequently linking to DBCO-derived QDs. The method is mild, reliable, and less destructive for the infectivity of viruses and the fluorescence of QDs since no Cu2+ is used. The labeled virions were so strong in fluorescence that single-virion imaging and tracking could be conveniently realized. The simple and universal labeling method has laid the foundation for longterm dynamic visualization of virus infection processes to understand the mechanism.

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ASSOCIATED CONTENT

S Supporting Information *

Two figures and two movies as described in the text. This material is available free of charge via the Internet at http:// pubs.acs.org.

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