3
-
Fig. 3. (A) Fluorescence responses of Cu(II)-BTPY (10 μM) to various RNS and ROS analysis substances (1 mM of HNO (Angelis` salt), NO, H
2 2
O , ClO ,
1
-
-
-
O , NO , NO , •OH and ONOO is the concentration of 0.1 mM, PBS buffer solution pH 7.4) in THF. F represents the initial maximum emission intensity of
2
2
3
0
Cu(II)-BTPY, F represents the final emission intensity of various ROS or RNS added to the probe Cu(II)-BTPY. The error bars represent the standard
deviation. (B) Fluorescence responses of Cu(II)-BTPY (10 μM) to HNO analysis substances (1 mM of HNO (Angelis` salt) in THF. Excited wavelength =
5
30 nm. (C) EPR spectra recorded at 298 K for 0.5 mM Cu(II)-BTPY in aqueous DMSO (blue line) and with excess Angelis` salt (red line).
4
-1
Ka equal to 3.2 ×10 M that was calculated by the Benesi-
Hildebrand equation (Fig. S2). In order to realize the fluorescent
‘
with Cu(II)-BTPY in DMSO accompanied by 5% CO flowing
2
at 37 °C for 30 minutes, and no change in emission intensity was
observed for cultures to which Angelis` salt was not added (Fig.
5 A, B). The following treatment of these cells with 3 mM
Angelis` salt led to an increment in fluorescence in red channel
(Fig. 5 C, D). The dramatic changes ascertain the potential
practicability of Cu(II)-BTPY for monitoring HNO in living
cells. In addition, we used MTS analysis to study the cytotoxicity
of different concentrations of Cu(II)-BTPY or BTPY via
incubation with HeLa cells for 24 hours. The cell viability of
Cu(II)-BTPY or BTPY was 87.2% or 89.3%, respectively (Fig.
S15, Fig. S16). Ultimately, zebrafish embryos were employed for
the staining study to prove the applicability of BTPY skeleton in
the more complex organism. As shown in Fig. 5 E-H, incubated
with 10 μM BTPY within 30 minutes, the imaged zebrafish
embryos display bright red fluorescence.
turn-on’ performance upon HNO, Cu(II)-BTPY complex was
prepared from the coordination of BTPY with CuCl (Scheme 1),
and certified by the positive ion electrospray mass spectrum
suggesting a peak with m/z = 915.2206 that conforms to that of
2
+
[
Cu(II)-(BTPY)Cl] (calcd m/z = 915.2235) (Fig. S14).
Also, the responses of Cu(II)-BTPY to the reductive
biological molecules were studied. Considering their importance
in biological studies, reactive oxygen species (ROS) and reactive
nitrogen species (RNS) (including NO, H O , ClO , O , NO ,
NO , •OH and ONOO ) were explored in comparison to HNO.
As expected, Cu(II)-BTPY shows high selectivity toward HNO
over relevant ROS and RNS species by measuring the respondent
changes in the optical spectra while gradually adding consecutive
amounts of ROS and RNS (Fig. 3A). Only the response of HNO
dis-plays the fluorescence enhancement in the Cu(II)-BTPY
solution, as reflected by the emission color of solution turned
from dark to bright pink (Fig 4). The fluorescence response of
-
1
-
2
2
2
2
-
-
3
-
1
-
-
others ROS and RNS such as NO, H O , ClO , O , NO , NO ,
OH and ONOO is negligible. The difference of HNO from
2
2
2
2
3
-
•
other ROS and RNS on Cu(II)-BTPY can be visually
distinguished under UV light (Fig. 3D). As profiled in Fig. 3B,
upon addition of 5 equiv HNO to Cu(II)-BTPY, the fluorescence
increased gradually to a maximum at 592 nm with a simultaneous
turned-on mode, implying that Cu(II)-BTPY was completely
reduced by HNO. A typical paramagnetism with a symmetric line
with g equal to 2.09 given by electron paramagnetic resonance
(EPR) measurement confirms the reduction of the Cu(II)-BTPY
Fig. 5. HNO-induced fluorescence response in HeLa cells: (A, B) Images of
cells stained with Cu(II)-BTPY (10 μM) in DMSO for 30 min at 37 °C. (C,
D) Cells were incubated with Cu(II)-BTPY in DMSO for 30 min and then
HNO (Angelis` salt) for 30 min at 37 °C. Scale bar, 50 μm; Fluorescence
images of probe BTPY in Zebrafish embryos: (E, F) Image of Zebrafish
embryos stained without BTPY (10 μm) in DMSO for 30 min at 37 °C. (G,
H) Image of Zebrafish embryos stained with BTPY (10 μM) in DMSO for 30
min at 37 °C. Scale bar, 200 μm. (A, C, E, G,) Bright field, (B, D, F, H) dark
field. HeLa Cells and Zebrafish embryos images were obtained using an
OLYMPUS IX71 inverted phase contrast fluorescence microscope.
into Cu(I)-BTPY. Excessively introducing Angelis` salt into the
Cu(II)-BTPY solution resulted in a rapid decrement in the EPR
signal that owes much to the diamagnetism of the reduced Cu(I)-
BTPY (Fig. 3C). The HNO sensing properties of Cu(II)-BTPY
were further probed by fluorescence titration, indicating that
Cu(II)-BTPY is an excellent candidate for highly selective turn-
on sensor in HNO identification.
Conclusions
In summary, we have described the design and synthesis of
the turn-on reaction sensor on the basis of BODIPY-terpyridine-
Cu(II) platform for highly selective detection of HNO over the
other ROS and RNS species. It is worth mentioning that the
relatively high fluorescence quantum efficiency and large stokes
shift of the sensor is useful for imaging experiments especially in
living organisms. On the other hand, a series of transition metal
Fig. 4. The photo of Cu(II)-BTPY under ultraviolet lamp (365nm) after
addition of different ROS and RNS.
Additionally, the biologically discernable ability of Cu(II)-
BTPY was evaluated via fluorescence imaging towards HNO
operated in cervical cancer HeLa cells. We incubated the cells
2+
ions were exploited, presenting Cu as the optimal quenching