SUPPLEMENTARY INFORMATION
Real-Time Tracking of Gold Nanoparticlesbioconjugated Respiratory Syncytial Virus
Infecting HEp-2 Cells
Xiao-Yan Wan, 1 Lin-Ling Zheng, 2 Peng-Fei Gao, 1 Xiao-Xi Yang, 1
Chun-Mei Li, 1 Yuan Fang Li 2 and Cheng Zhi Huang 1 , 2
1Key
Laboratory of Luminescence and Real-Time Analytical Chemistry
(Southwest University), Ministry of Education, College of Pharmaceutical
Sciences, Southwest University; 2College of Chemistry and Chemical
Engineering, Southwest University, Chongqing 400715, China
Supplementary methods
Preparation of AuNPs. Monodispersed AuNPs of varying sizes were prepared by
altering the quantity of HAuCl4 and reducing agents. For the preparation of AuNPs of
6.5 nm, it was made according to the previous report with slight modification. 1 2 mL
HAuCl4 (1%, w/w) and 48 mL H2O were heated together with magnetic stirring. Then
Corresponding
author. Tel: +86-23-68254659;
chengzhi@swu.edu.cn (C.Z. Huang).
Fax:
+86-23-68367257.
E-mail:
the solution containing 0.5 mL sodium citrate (8%) and 225 μL tannic acid (4%) was
added quickly when beginning to boil. After refluxing for 5 min, turn off the heat and
allow the solution to cool to room temperature under stirring.
For 30 nm and 50 nm size of AuNPs, the synthesis steps were almost the same as
AuNPs of 13 nm with different amount of reagents and refluxed time. Both 30 and 50
nm of AuNPs were prepared by 0.5 mL HAuCl4 (1%, w/w) and 49.5 mL H2O, with
625 μL 1% sodium citrate with refluxing for 20 min, and 500 μL 1% sodium citrate
with refluxing for 15 min were used, respectively.
Biotin-RSV Inducing Syncytium Formation Investigation. RSV or biotin-RSV
was incubated in HEp-2 cells at a multiplicity of infection (MOI, ratio of infectious
virus particles to cells) of 5, respectively. After 2 h of incubation at 37 oC, the
inoculum was resplaced by prewarmed RPMI 1640 culture medium containing 2%
FBS and incubated for 24 h. Then optical images of HEp-2 cells was used to observe
syncytium formation.
Confocal Microscopy Imaging. HEp-2 cells were cultured on 12-well plates of
coverslip for about 24 h. Then 100 μL biotinylated viruses and 100 μL PBS buffer
were successively added to 12-well plates and incubated for 20 min at 4 oC, spare
virus solution was removed and cells in 12-well plates were rinsed. 400 μL PBS
buffer containing 0.4 μL QDs were added and incubated for 30 min at 4 oC. Then
cells were fixed with 4% paraformaldehyde, sealed with glycerin, and then transferred
for fluorescence imaging under a DSU live-cell confocal microscope (Olympus,
Japan) system with laser excitations of F380.
Immunofluorescence. Cells were incubated with Au-RSVs or RSVs at 4 oC for 30
min. After washing with PBS to remove the excess AuNP-RSVs/RSVs, infected cells
were culturing at 37 oC for different time (0.5, 1.0, 1.5 and 2.0 h). Then cells were
fixed in 4% (w/v) paraformaldehyde for 30 min. After washing with PBS, cells were
exposed 2% (w/v) BSA at 37 oC for 1 h. Cells were further incubated with
anti-Respiratory Syncytial Virus G glycoprotein antibody (Abcam) for 1.5 h at 37 oC,
followed by incubating with DyLight 488 conjugated goat antimouse IgG antibody
(Thermo scientific) for 1 h at 37 oC. Between two successive steps, the samples were
rinsed three to four times with PBS. The fluorescence images were acquired by a
DSU live-cell confocal microscope (Olympus, Japan).
Figure S1. RSV or biotin-RSV inducing syncytium formation investigation. The control group
has only HEp-2 cells (a). The images of syncytium are from the infection of RSV (b) and
biotin-RSV (c), respectively. Black arrows indicate the syncytium. The scale bars indicate 20 μm.
Figure S2. Confocal Microscopy Imaging of viral infection. The control group is represented by
HEp-2 cells (a). HEp-2 cells were incubated with QDs (b) and QD-RSVs (c). The scale bars
indicate 20 μm.
Figure S3. Immunofluorescence image of AuNP-RSVs and RSVs. Cells were incubated with
Au-RSVs or RSVs at 4
oC
for 30 min. After washing with PBS, cells infected with
AuNP-RSVs/RSVs were cultured at 37 oC for different time (0.5, 1.0, 1.5 and 2.0 h). Then, the
AuNP-RSVs/RSVs were immunostained with the mouse monoclonal antibody against the G
glycoprotein of RSV and DyLight 488 conjugated goat antimouse IgG (green). And the images
were acquired by the merge of bright field and fluorescence. The scale bars indicate 20 μm.
Reference
1 Xu, X.-H. N., Huang, S., Brownlow, W., Salaita, K. & Jeffers, R. B. Size and
Temperature Dependence of Surface Plasmon Absorption of Gold Nanoparticles
Induced by Tris(2,2‘-bipyridine)ruthenium(II). J. Phys. Chem. B 108,
15543-15551 (2004).