ChemComm
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Journal Name
COMMUNICATION
this study we decided to perform in vitro detection of This work was financially supported by The CAMS Innovation
superoxide anion in DMSO. Probe 1 can detect O2•− sensitively Fund for Medical Sciences (2017-I2DMO-I1: 1-00.11003)9, /DT0hCeC02D2r8u2Ag
in DMSO with the LOD of 1.1 μM.
Innovation Major Project (2018ZX09711001).
Secondly, special attention should be given to the solvents and
reaction buffers used in the ROS detection. For the detection of
ClO-, phosphate buffer is preferred because both Tris-HCl and
HEPES buffers can consume ClO-.9, 20, 21 In addition, DMSO is also
reported as a good ClO- scavenger, which can inhibit the
oxidation ability of ClO- at concentration as low as 0.00005%
(v/v).22 Thus, in the detection of ClO-, DMSO cannot be used to
prepare the stock solution of the probes. An interesting work by
Sando’s group described the oxidation between 7-amino-4-
methyl-3, 4-dihydrocoumarin and the ClO- in phosphate buffer
(pH 7.4) with 0.1% DMF.19 In this study, we also observed a
weak fluorescence turn-on between probe 1 (7-diethylamino-4-
methyl-3, 4-dihydrocoumarin) and excessive ClO- in phosphate
buffer (pH 7.4) with 0.1% DMF. However, if we used other
of probe 1, we cannot observe any obvious fluorescence turn-
on between probe 1 and excessive ClO- in phosphate buffer (pH
7.4) (ESI, Fig. S7). Thus, without the presence of the catalyst,
probe 1 does not react with ClO- in phosphate buffer.
Conflicts of interest
There are no conflicts to declare.
Notes and references
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Moreover, we also used these six stock solutions of probe 1 to
re-evaluate the reactions between probe 1 and several other
ROS. Our results showed that generally all six solvents (final
concentration 0.1%) did not affect the reaction between probe
ONOO-) in phosphate buffer (pH 7.4) (ESI, Fig. S8). We also
found that probe 1 in all six solvents can be oxidized by O2•− in
DMSO, and the reaction mixture exhibited the fluorescence
when diluted in various buffers (ESI, Fig. S9-11). This proved the
general application of probe 1 to detect superoxide anion.
13. B. Tang, L. Zhang and L. L. Zhang, Anal Biochem, 2004, 326,
176-182.
Lastly, we also synthesized probe 3 with enhanced hydrophobic
properties. Probe 3 was planned to be used in cellular level of
ROS examination. Unfortunately, we were unable to use either
14. W. Zhang, P. Li, F. Yang, X. Hu, C. Sun, W. Zhang, D. Chen
and B. Tang, J Am Chem Soc, 2013, 135, 14956-14959.
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probe 1 or its’ derivative 3 (ESI) to sense the O2 produced in
•−
PMA treated mammalian cells.14, 16 Considering O2 is placed
the most top position for its oxidability in all ROS,1, 2 selective
•−
O2 probes usually exhibit low reactivity, which will not react
with other ROS. In this study, probe 1 and its derivative 3 can
•−
selectively recognize O2 but not other ROS, thus the overall
reactivity of probe 1 and 3 should be low, and we speculated
only a marginal amount of probe 3 was oxidised by relatively
low level of O2•− in the complex cellular environment.
19. T. Doura, H. Nonaka and S. Sando, Chem Commun (Camb),
2012, 48, 1565-1567.
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H. Li, J. Chen, W. Wang and S. Sun, Chem Commun (Camb),
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22. L. M. Kabeya, M. F. Andrade, F. Piatesi, A. E. Azzolini, A. C.
Polizello and Y. M. Lucisano-Valim, Anal Biochem, 2013,
437, 130-132.
In conclusion, we report that the use of 7-diethylamino-4-
methyl-3, 4-dihydrocoumarin as a reaction based fluorescent
probe to selectively detect O2•− in solution. In addition, we also
proposed and proved the advantages of in vitro detect O2 in
anhydrous DMSO instead of aqueous buffers when used KO2 as
the source of superoxide anion.
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