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dimer connected by an alkene bond, forming the binuclear
ruthenium(II) dimer (BiRD) as shown in Fig. 1B. Alkene bonds
are known to be labile to oxidative agents.5 We generated the
alkene-linked BiRD by first dimerizing 5phen using a fumaryl
linker. The dimeric 5phen was then converted to the BiRD with
cis-dichlorobis(2,20-bipyridine) Ru(II) in a similar method as the
monomer. The identity of the BiRD was confirmed with mass
spectrometry and NMR. The absorption spectra of the mono-
mer and BiRD are shown in Fig. S1 (ESI†).
Photosensitizer dimers are able to self-quench with respect
to fluorescence and singlet oxygen generation when linked in
close proximity.6 The fluorescence of the absorbance-matched
BiRD was attenuated approximately 20 fold compared to the
monomeric Ru(II) complex, as shown in Fig. 1C. Generation of
singlet oxygen was next assessed using the method of chemical
bleaching of the p-nitrosodimethylaniline (RNO) chromo-
phore.7 In this assay, histidine is used as an intermediate
which reacts with the singlet oxygen to generate a trans-
annular peroxide which can react with other species including
the RNO indicator. As shown in Fig. 1D, using absorbance-
matched Ru(II) complexes, upon irradiation with a blue 450 nm
excitation laser, singlet oxygen generation was significantly
attenuated in the BiRD compared to the monomer. Histidine
was required to achieve bleaching of the RNO. Thus, the BiRD
displayed the expected properties of attenuated fluorescence
and singlet oxygen generation.
Fig. 2 BiRD activation via irradiation of another photosensitizer. (A) Absorp-
tion spectra of the BiRD and methylene blue. The laser excitation wavelengths
corresponding to the excitation of the two dyes are indicated. (B) Fluores-
cence activation kinetics (assessed with 450 nm excitation and 600 nm
emission) of the BiRD (50 mM) by methylene blue (20 mM) using irradiation
with a red laser (300 mW) in D2O. (C) Control experiments of BiRD activation
by methylene blue in D2O. Concentrations used were 50 mM BiRD, 1 mM
histidine and 20 mM MB. Both laser fluences were 810 J cmꢁ2 from lasers
outputting 300 mW and irradiating the samples for 15 minutes. Mean ꢀ std
dev. for n = 3.
The BiRD was next assessed for activation following expo-
sure to photosensitization by a second photosensitizer. To
avoid directly exciting the BiRD, a longer wavelength photo-
sensitizer was selected, since photosensitizers can generally
only be excited at wavelengths shorter than their emission.
Next, the BiRD was used to develop a chemical amplification
As shown in Fig. 2A, the commonly used photosensitizer scheme. As shown in Fig. 3, conventional detection of photo-
methylene blue was used since it has a red-shifted absorption sensitizers using a chemical bleaching assay involves the single
peak around 670 nm. In D2O, methylene blue has a singlet step of irradiating the photosensitizer in the presence of an
oxygen quantum yield of 0.52,8 whereas the Ru(II) monomer indicator such as RNO (route I). By using a BiRD activation
singlet oxygen quantum yield was determined to be 0.16 (see intermediate step as shown in route II, a chemical amplifica-
ESI†). However, at the methylene blue excitation wavelength, tion system is created. The efficacy of the system is based on the
the BiRD exhibited negligible excitation. The spectral location fact that the initial photosensitization step in IIa need not
of the blue laser used to excite the BiRD and the red laser used bleach an indicator dye that itself need be detected, but rather
to excite methylene blue were separated by over 200 nm. As only must activate the BiRD photosensitizer. The BiRD photo-
shown in Fig. 2B, when methylene blue and the BiRD were sensitizer, is then irradiated to initiate a second step of
incubated together and exposed to the red laser, an 8 fold chemical bleaching of an indicator dye (route IIb). 10 nM
restoration in the BiRD fluorescence was achieved. Without the methylene blue was not a sufficient concentration to directly
presence of methylene blue, direct irradiation of the BiRD with induce significant bleaching of the RNO indicator upon irra-
the red laser had no effect on BiRD activation. As shown in diation with a red laser (route I). This was not surprising, since
Fig. 2C, both histidine and methylene blue were required to photosensitizers are usually assessed at concentrations that are
achieve BiRD activation. Histidine likely plays the same role as orders of magnitude higher (in the micromolar range). How-
in RNO bleaching in trapping the singlet oxygen in a reactive trans- ever, under the same irradiation conditions, 10 nM methylene
annular peroxide. A photocleavage product from activation of BiRD blue could effectively activate the BiRD, as indicated by the
could be directly observed using HPLC (Fig. S2, ESI†). The smaller increase in the BiRD fluorescence. The activated BiRD was then
mass of the emerging peak was consistent with the assumption irradiated with a blue laser and resulted in the effective
that the BiRD is cleaved at the alkene bond. However, further work bleaching of the RNO indicator. Thus, using an intermediate
is required to accurately elucidate the cleavage mechanism. But chemical amplification effect with a ROS-sensitive photosensi-
clearly the BiRD could selectively be activated by a longer wave- tizer offered improvement over conventional methodology.
length photosensitizer. The BiRD demonstrated activation by sub-
nanomolar levels of methylene blue (Fig. S3, ESI†).
In summary, we have developed a BiRD that is, to our
knowledge, the first photosensitizer reported that itself is activated
3232 | Chem. Commun., 2014, 50, 3231--3233
This journal is ©The Royal Society of Chemistry 2014
Lou, Kun
