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Luminescent gold nanocluster-based sensing platform for accurate H2S detection in vitro and in vivo with improved anti-interferenceQi Yu1, Pengli Gao1, Kenneth Yin Zhang1, Xiao Tong1, Huiran Yang1, Shujuan Liu1, Jing Du1, Qiang Zhao1 and Wei Huang1,2
1 Key Laboratory for Organic Electronics and Information Displays and Institute of Advanced Materials, Nanjing University of Posts and Telecommunications, Nanjing 210023, P. R. China; 2 Key Laboratory of Flexible Electronics and Institute of Advanced Materials, Jiangsu National Synergetic Innovation Center for Advanced Materials, Nanjing Tech University, Nanjing 211816, P. R. China.Correspondence: E-mail: [email protected] (Q Zhao); E-mail: [email protected] (W Huang)
Synthesis of MUA-Capped Au NCs
MUA (13.1 mg) and NaOH (200 L, 1 M) were added into 20 mL of H2O. HAuCl4
(510 L, 1% by mass) and a tetrakis(hydroxymethyl)phosphonium chloride (THPC)
(445 L) solution were added into the mixture. The THPC solution was prepared by
adding an 80% THPC solution (12 L) to water (1 mL). The mixture was stirred for 2
h. Au NCs@MUA was obtained.
Synthesis of Au NCs@Chi
The Au NCs (2 mg) were dispersed in 800 L of water. 1-Ethyl-3-(3-
(dimethylamino)propyl)carbodiimide (EDC) (17.6 mg) and N-hydroxysuccinimide
(NHS) (3.5 mg) were added into the solution. The pH value was adjusted to 5, and the
mixture was stirred for 4 h. After 4 h, a Chi (average molecular weight, 5000) solution
(1.0 mg/mL) was added to the mixture. The solution was stirred for 20 h and
separated by centrifuging at 20000 rpm for 40 min. The obtained Au NCs@Chi was
dispersed in 2 mL of water. To investigate the effect of the concentration of Chi,
various concentrations of Chi (0.5, 1.0, 2.0 and 5.0 mg/mL) were used for the
preparation. Additionally, we used the same method to obtain Au NCs@Chitosan
with a varied molecular weight. The average molecular weights of chitosan used were
50-190, 190-310 and 310-375 kDa.
Synthesis of Au NCs-Cy1
Modification of Cy1 on Au NCs@Chi was also performed by EDC/NHS chemistry.
Cy1 (2 mM), EDC (16 mg) and NHS (23 mg) were added to 2 mL of ethanol, and the
mixture was stirred for 2 h. Freshly prepared Au NCs@Chi was dispersed in 2 mL of
water and added to the mixture. The solution was stirred for 20 h, and Au NCs-Cy1
was separated by centrifuging at 20000 rpm for 40 min in a 1:1 ethanol and water
mixture. To investigate the effect of the concentration of Cy1, various concentrations
of Cy1 (2, 20 and 200 M) were used for the preparation of Au NCs-Cy1.
Figure S1. EDS analysis of MUA capped Au NCs.
Table S1 Quantum yields of Au NCs@Chi with varied concentration of Chi.
0.5 mg/mL 1.0 mg/mL 2.0 mg/mL 5.0 mg/mL
Quantum yields 7% 7% 9% 10%
0.0
0.4
0.8
1.2
I/I
0 Au NCs Au NCs@Chi
0.0
0.4
0.8
1.2
450 500 550 600 6500
50
100
150
PL In
tens
ity (a
.u.)
Wavelength (nm)
Au NCs@Chi + 1 mM GSH
450 500 550 600 6500
50
100
150
PL In
tens
ity (a
.u.)
Wavelength (nm)
Au NCs@Chi Au NCs@Chi in DMEM
a b
450 500 550 600 6500
200
400
600
800
PL In
tens
ity (a
.u.)
Wavelength (nm)
Au NCs + 1 mM GSH
c
d
450 500 550 600 6500
50
100
150
200
250
300
PL In
tens
ity (a
.u.)
Wavelength (nm)
Au NCs Au NCs in DMEM
e f
4 5 6 7 8 9
0.6
0.8
1.0
1.2
4 5 6 7 8 9
0.6
0.8
1.0
1.2
Au NCs
I/I0
pH
Au NCs@Chi
I/I0
pH
Figure S2. (a) Relative luminescence (I/I0) of aqueous Au NCs (black) and Au NCs@Chi (red) solution (25 g/mL) in the presence of 1 mM of various anions; (b) Relative luminescence (I/I0) of aqueous Au NCs (black) and Au NCs@Chi (red) solution(25 g/mL) dispersed in PBS buffer with different pH values; PL spectra of AuNCs (c) and AuNCs@Chi (d) in PBS buffer and PBS buffer containing 1 mM GSH; PL spectra of AuNCs (e) and AuNCs@Chi (f) in PBS buffer and cell culture, DMEM. ex = 405 nm.
Figure S3. PL spectral traces of Au NCs (25 g/mL) in PBS buffer (pH = 7.4) in the presence of 0 1 mM NaHS. ex = 405 nm.
300 400 500 600 700300 400 500 600 700300 400 500 600 700
PL
Inte
nsity
(a.u
.)
Wavelength (nm)
PL Cy1
PL
Inte
nsity
(a.u
.)
Wavelength (nm)
PL Au NCs A
bs.
Wavelength (nm)
Abs. Cy1
Abs. Au NCs
Figure S4. Normalized absorption spectra of Au NCs (black) and compound Cy1 (blue), normalized PL spectra of Au NCs (red) and compound Cy1 (green) (ex = 405 nm).
Figure S5. Normalized absorption and PL spectra of Au NCs-Cy1 (25 g/mL) in PBS buffer (pH = 7.4).
0 200 400 600 8000.0
5.0x103
1.0x104
1.5x104
K
inet
ic S
can
Time (s)0 200 400 600 800
0.0
5.0x103
1.0x104
1.5x104
Kin
etic
Sca
n
Time (s)
ba
Figure S6. Photostability experiments of Au NCs-Cy1 (25 g/mL) monitored at 512 and 604 nm with a xenon lamp excitation source. ex = 405 nm.
0 200 400 600 8000
1x104
2x104
K
inet
ic s
can
Time (s)0 200 400 600 800
1.76x104
1.87x104
1.98x104
2.09x104
Kin
etic
sca
n
Time (s)
Figure S7. Photostability experiments of Au NCs and Au NCs@Chi with a xenon lamp excitation source. ex = 405 nm.
0.0
0.1
0.2
0.3
0.4
0.5
0.6
Rat
io (I
512/
604)
0.0
0.1
0.2
0.3
0.4
0.5
Rat
io (I
512/
604)
K+ Na+ Ca2+ Fe3+ Cu2+ Zn2+ Mg2+
Figure S8. The luminescence ratio of Au NCs-Cy1 in the presence of different ions (1 mM).
Figure S9. Ratiometric luminescence changes of Au NCs-Cy1 (25 g/mL) in the presence of various amounts of NaHS in PBS buffer (pH = 7.4). ex = 405 nm.
Freshly prepared
1 10 100 1000
Num
ber
Diameter (nm)
After 15 days
Freshly prepared
1 10 100 1000
Num
ber
Diameter (nm)
After 15 days
Freshly prepared
1 10 100 1000
Num
ber
Diameter (nm)
After 15 days
0.5 mg/mL
2.0 mg/mL 5.0 mg/mL
Freshly prepared
1 10 100 1000
Diameter (nm)
Num
ber
After 15 days
1.0 mg/mL
Figure S10. DLS of Au NCs@Chi that were freshly prepared and stored after 15 days with varied concentration of Chi (0.5, 1.0, 2.0 and 5.0 mg/mL).
200 nm50 nm 50 nm
100 nm 200 nm 200 nm
a. b. c.
d. e. f.
Figure S11. TEM images of Au NCs@Chi that used 0.5 mg/mL (a), 2.0 mg/mL (b), 5.0 mg/mL (c) Chi. TEM images of Au NCs@chitosan that used chitosan with average mol weight 50-190 kDa (d), 190-310 kDa (c) and 310-375 kDa (d).
Figure S12. Normalized PL spectra of Au NCs@Chi with varied concentration of
Chi.
Figure S13. The luminescence ratio of Au NCs-Cy1 that was functionalized with different concentrations of Chi in the absence and presence of NaHS (100 M).
Freshly prepared
1 10 100 1000
Num
ber
Diameter (nm)
After 15 days
Freshly prepared
1 10 100 1000
Num
ber
Diameter (nm)
After 15 days
Freshly prepared
1 10 100 1000
Num
ber
Diameter (nm)
After 15 days
Freshly prepared
1 10 100 1000
Diameter (nm)
Num
ber
After 15 days
a.
c. d.
b.
Figure S14. DLS of Au NCs@chitosan that were freshly prepared and stored after 15 days with varied average mol weight of chitosan (a. 5000, b. 50-190 kDa, c. 190-310 kDa, d. 310-375 kDa).
Figure S15. Normalized PL spectra of Au NCs@chitosan with varied average mol weight of chitosan.
Table S2 Quantum yields of Au NCs@chitosan with varied average mol weight of chitosan.
5000 50-190 kDa 190-310 kDa 310-375 kDa
Quantum yields 7% 9% <1% <1%
Figure S16. The luminescence ratio of Au NCs-Cy1 that was functionalized with different average mol weight of chitosan in the absence and presence of NaHS (100 M).
0 5 10 15 20 250.4
0.8
1.2
1.6
Rat
io
CNaHS (M)0 10 20 30 40
1.0
1.1
1.2
Rat
io
CNaHS (M)0 5 10 15 20
1.0
1.2
1.4
Rat
io
CNaHS (M)
Figure S17. The luminescence ratio of Au NCs-Cy1 with varied concentration of Cy1 in the presence of NaHS in PBS buffer (pH = 7.4).
0.4
0.8
1.2
1.6
I 512
/I 604
90 s 5 min 10 min
500 600 7000.0
2.0x103
4.0x103
6.0x103
PL
Inte
nsity
(a.u
.)
Wavelength (nm)
0 s 90 s
0 20 40 60 80 100 1200.3
0.6
0.9
1.2
1.5
1.8
I 512/I
604
Time (s)
a b
Figure S18. (a) Time-dependent ratiometric luminescence changes of Au NCs-Cy1 (25 g/mL) in the presence of NaHS (100 M) in PBS buffer (pH = 7.4). Inset is the PL spectra of Au NCs-Cy1 in the absence of NaHS and in the presence of NaHS (100 M) after 90 s. (b) Ratiometric luminescence changes of Au NCs-Cy1 in the presence of 100 M NaHS and other biologically relevant competing analytes in PBS buffer.
Table S3 Decay times and normalized pre-exponentials of Au NCs-Cy1 at different HS concentrations. PL decay traces collected at ex = 375 nm and em = 512 nm.CHS
(M) 1 (ns) (f1) 2
(ns) (f2) 3 (ns) (f3) ave(ns)
0 0.70 (87.7%) 7.00 (7.9%) 40.3 (4.4%) 25.6
10 2.60 (18.6%) 18.2 (43.4%) 86.1 (38.0%) 41.0
20 4.10 (16.8%) 21.6 (47.2%) 92.2 (36.0%) 44.1
35 4.60 (17.5%) 27.9 (40.6%) 130 (41.9%) 66.4
50 5.40 (21.8%) 36.5 (45.3%) 153 (33.0%) 68.3
75 10.0 (14.2%) 54.1 (41.4%) 222 (44.7%) 123
Table S4 Decay times and normalized pre-exponentials of Au NCs-Cy1 at different HS concentrations. PL decay traces collected at ex = 375 nm and em = 604 nm.CHS
(M) 1 (ns) (f1) 2
(ns) (f2) 3 (ns) (f3) ave (ns)
0 3.00 (42.8%) 16.8 (39.8%) 93.2 (17.4%) 24.2
10 3.20 (28.0%) 18.4 (40.0%) 118 (31.9%) 46.0
20 3.70 (28.0%) 22.4 (42.8%) 131 (29.2%) 49.0
35 3.10 (23.5%) 31.9 (40.3%) 155 (36.2%) 69.8
50 4.40 (29.3%) 35.7 (40.2%) 219 (30.4%) 82.2
75 4.70 (11.9%) 42.0 (34.7%) 299 (53.5%) 175
0.6
0.9
1.2
1.5
1.8
HbThroLysBSA Pepsi
Rat
io (I
512/
I 604
)
Try
-HS-
+HS-
a b
0.4
0.8
1.2
1.6 -HS-
+HS-
Mg2+Zn2+Cu2+Ca2+K+ Na+
Rat
io (I
512/
I 604
)
Fe3+
0.4
0.8
1.2
1.6
2.0
Mg2+Zn2+Cu2+Fe3+Ca2+
Rat
io (I
512/
I 604
)
-HS-
+HS-
c
Figure S19. The luminescence ratio of Au NCs-Cy1 in the presence of different ions (10-3 M) (a) and (10-7 M) (b). (c) The luminescence ratio of Au NCs-Cy1 in the presence of BSA, pepsin, trypsin, lysozyme, thrombin and hemoglobin.
Green Channel
Red Channel
a b c d
1 2 3 40
4000
8000
12000
16000
20000
Green channel Red channel Ratio
1 2 3 40.0
0.2
0.4
0.6
0.8
1.0
e
Figure S20. Evaluation of uptake mechanism of Au NCs-Cy1 in HeLa cells under different conditions: (a) cells were incubated with Au NCs-Cy1 (25 g/mL) for 2 h at 37 C; (b) cells were incubated with Au NCs-Cy1 for 2 h at 4 C; (c) cells were preincubated with 50 mM 2-deoxy-D-glucose and 5 M oligomycin for 1 h and then incubated with Au NCs-Cy1 at 37 C for 2 h; (d) cells were preincubated with 50 mM NH4Cl for 1 h and then incubated with Au NCs-Cy1 at 37 C for 2 h; (e) Luminescence intensity of HeLa cells recorded from the green channel (black) and the red channel (red) and the intensity ratio Green/Red (blue) under different conditions: (1) cells were incubated with Au NCs-Cy1 for 2 h at 37 C; (2) cells were incubated with Au NCs-Cy1 for 2 h at 4 C; (3) cells were preincubated with 50 mM 2-deoxy-D-glucose and 5 M oligomycin for 1 h and then incubated with Au NCs-Cy1 at 37 C for 2 h; (4) the cells were preincubated with 50 mM NH4Cl for 1 h and then incubated with Au NCs-Cy1 at 37 C for 2 h. These results were obtained by flow cytometry analysis.
h i
Green channel
Red channel
gfe
a b c d
Green Channel
Red Channel
Ratio
0.00
1.05
1.87
2.72
3.62
Igreen/Ired
Figure S21. Confocal laser scanning microscopy and ratiometric luminescence images of HeLa cells under different conditions: (a) Cells were incubated with Au NCs-Cy1 (25 g/mL) at 25 °C in the presence of NaHS (100 M); (b) cells were treated with Au NCs-Cy1 (10 g/mL) for 2 h in the presence of NaHS; (c) the time of incubation was elongated to 4 h in the presence NaHS; (d) the laser power was randomly increased in the presence of NaHS. Flow cytometry analysis results of HeLa cells. (e) blank cells; (f) Cells were incubated with Au NCs-Cy1 (25 g/mL) at 25 °C in the presence of NaHS; (g) cells were treated with Au NCs-Cy1 (10 g/mL) for 2 h in the presence of NaHS; (h) the time of incubation was elongated to 4 h in the presence of NaHS; (i) the laser power was randomly increased in the presence of NaHS.
30 m
Fast lifetime (ns)
110
0
a b
c d
Figure S22. PLIM images of live HeLa cells under different conditions. (a) Cells were incubated with Au NCs-Cy1 (25 g/mL) at 25 °C in the presence of NaHS (100 M); (b) cells were treated with Au NCs-Cy1 (10 g/mL) for 2 h in the presence of NaHS; (c) the time of incubation was elongated to 4 h in the presence of NaHS; (d) the laser power was randomly increased in the presence of NaHS. Excitation wavelength was 405 nm. Lifetimes were collected through 520 20 nm bypass.
Charaterization of compound Cy11H NMR spectrum
N
HOOC
N
Br
13C NMR spectrum
N
HOOC
N
Br
MALDI-TOF mass spectrum
N
HOOC
N
Br
