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Electronic Supplementary Information (ESI)
Two-Color-Based Nanoflares for Multiplexed MicroRNAs Imaging in
Live Cells
Jing Li1, Jin Huang*1, Xiaohai Yang1, Yanjing Yang1, Ke Quan1, NuliXie1, Yanan Wu1,
Changbei Ma2 and Kemin Wang*1
1State Key Laboratory of Chemo/Biosensing and Chemometrics, College of
Chemistry and Chemical Engineering, Key Laboratory for Bio-Nanotechnology and
Molecular Engineering of Hunan Province, Hunan University, Changsha, China.
2 State Key Laboratory of Medical Genetics & School of Life Science, Central South
University, Changsha, China.
Corresponding Author * E-mail: [email protected]. * E-mail: [email protected].
Fig. S1. TEM image of AuNPs
Fig. S2. Absorption spectra of AuNPs (black line) and DNA functionalized
AuNPs (red line). The maximum optical absorption was shifted from 519 nm to
524 nm after modification.
Wavelength (nm)400 450 500 550 600 650 700
Abs
orpt
ion
0.0
.1
.2
.3
.4
.5
.6
AuNPsNanoflares
Fig. S3. The fluorescence recovery of different length flares (11, 12, 13, 14,
15bases) at various temperatures (red ring represents fluorescence recovery at
37 oC). The fluorescence of FAM was excited at 488 nm and measured at 520
nm.
Fig. S4. the kinetic analysis of fluorescence intensity on the hybridization time for
Nanoflares incubated with miRNA-21 target. The fluorescence of FAM was excited at
488 nm and measured at 520 nm.
Time (min)0 10 20 30 40 50 60 70
Fluo
resc
ence
inte
nsity
(a.u
.)
0
100
200
300
400
500
600
700no target target
Fig. S5. Evaluation of Amounts of DNA Duplexes on each AuNP. Standard
linear calibration curve of fluorescence signal against the concentration of FAM
labeled flares-21 a) and Cy5 b) labeled flare.
Concentration (nM)0 20 40 60 80 100 120 140 160
Flu
ores
cenc
e in
tens
ity
(a.u
.)
0
1000
2000
3000
4000
5000
Wavelength (nm)500 520 540 560 580 600 620 640 660
Fluo
resc
ence
inte
nsity
(a.u
.)
0
1000
2000
3000
4000
5000
Concentration (nM)0 20 40 60 80 100 120 140 160
Flu
ores
cenc
e in
tens
ity
(a.u
.)
-100
0
100
200
300
400
500
600
700
Wavelength (nm)640 660 680 700 720 740 760
Flu
ores
cenc
e in
tens
ity
(a.u
.)
0
100
200
300
400
500
Fig. S6. Nuclease stability of the nanoflares in the presence or absence of DNase I.
Fluorescence curves of the nanoflares (3 nM) without (a) or with (b) DNase I for 1h.
Insets: fluorescence spectra after hybridization of the nanoflares with DNA targets in
the absence (c) and presence (d) of DNase I. The above is miRNA-21 DNA target
measured at 488 nm. The below is miRNA-141 DNA target measured at 635 nm.
Time (min)0 10 20 30 40 50 60 70
Fluo
resc
ence
inte
nsity
(a.u
.)
0
500
1000
1500
2000
Nanoflares/DNase INanoflares
Wavelength (nm)500 520 540 560 580 600 620 640 660
Fluo
resc
ence
inte
nsity
(a.u
.)
0
100
200
300
400
500
600
700
Nanoflares /DNase INanoflares
Time (min)0 10 20 30 40 50 60 70
Fluo
resc
ence
inte
nsity
(a.u
.)
40
60
80
100
120
140
160
180
200
Nanoflares/DNaseINanoflares
Wavelength (nm)640 660 680 700 720 740 760
Fluo
resc
ence
inte
nsity
(a.u
.)
0
50
100
150
200
250
300
350
Nanoflares/DNase INanoflares
Fig. S7. Growth inhibition assay (MTT). LOVE-1 cells were incubated with
unmodified AuNPs (1 nM), nanoflares (1 nM and 5 nM) for 6 h, 12 h, 24 h and 48 h.
Blank bar stands for the unmodified Au NPs; red bar stands for the nanoflares (1 nM);
green bar stands for higher concentration of the nanoflares (5 nM).
Fig. S8. Confocal images of four cells treated with nanoflares for 2,4,6,8 and 10 h at
37 oC, 7721 and 22Rv1 cells were chosen as the control cells of Cy5 and FAM signal,
respectively. The red fluorescence signals were recorded using Cy5 in the red channel
with 633 nm excitation and the green fluorescence signals were recorded using FAM
in the green channel with 488 nm excitation. Scale bar = 10 µm.
2h 4h 6h 8h
LOVE-1
10h
7721
HeLa
2h 4h 6h 8h 10h
22Rv1
Fig. S9. Flow cytometry analysis of various cells type treated with nanoflares.
Fluorescence intensity
Cou
nt
FAM
Fluorescence intensity
Cou
nt
Cy5
gene
Sample
miRNA-14
1 U6 ∆Ct ∆∆Ct 2 -(∆∆Ct)
1 24.18793 12.85328 11.33464 0 1
2 29.4299 13.83117 15.59873 4.264091 0.052045
3 24.91745 14.24484 10.67261 -0.66203 1.582311
4 29.57694 14.77521 14.80173 3.467093 0.090428
Gene
Sample miRNA-21 U6 ∆Ct ∆∆Ct 2 -(∆∆Ct)
1 23.83093 12.85328 10.97765 0 1
2 24.08488 13.83117 10.25372 -0.72393 1.651675
3 22.84368 14.24484 8.598843 -2.37881 5.201058
4 23.26503 14.77521 8.489822 -2.48783 5.609318
Fig. S10. The expression analysis of miR-21 and miR-141 in four different cells
respectively by qRT-PCR. Real-time fluorescence curves and relative expression level
of miRNA-21and miRNA-141 in cells by qRT-PCR analysis.
