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solvents (Supporting Information, Figure S6), whereas similar
properties in DMSO/water solutions were also observed (Sup-
porting Information, Figures S7 and S8). However, it is worth
noting that the aggregation tendency of dU-SQ was much
stronger than 5 in DMSO/water mixtures, as evidenced by their
absorption spectra; for example, 50% water can lead to obvi-
ous H-aggregation of dU-SQ but 80% water was needed to
trigger the same behavior in 5. This is likely due to the more
planar structures and extended p-systems after conjugating
with the deoxyribonucleosides.
rotation.[3] For normal “molecular-rotor-mechanism”-based vis-
cosity sensors, their viscosity-dependent fluorescence intensity
can be described by the Fçrster–Hoffmann equation.[23] Even
though aggregation effects are added to the “molecular-rotor
mechanism” for deoxyribonucleoside-modified squaraines in
glycerol/water solvents, log–log analysis of the emission inten-
sity data as a function of solvent viscosity generated accepta-
ble linear relationships for dU-SQ with R2 =0.93 and dA-SQ
with R2 =0.94 (Supporting Information, Figures S10 and S11).
dU-SQ was later selected to perform viscosity-sensing ex-
periments at the cellular level because of its higher fluores-
cence compared with dA-SQ. Since the biological environment
is complex within cells, and some squaraine structures have
shown enhanced emission upon addition of serum albumin[24]
and lipid membranes,[4b,25] studies of the interaction of dU-SQ
with biomolecules were performed in the presence of calf
thymus DNA, bovine serum albumin, and biomimetic condi-
tions (e.g., liposomes and pluronic micelles), and none of them
resulted in a significant change in the optical properties of the
dye (Supporting Information, Figure S12).
In addition, because of the highly hydrophobic zwitterion
structure, 5 was insoluble in water and precipitation occurred
in glycerol/water mixtures, which hampered further biological
applications. However, after chemically linking the squaraine to
hydrophilic deoxyribonucleosides, the squaraine analogs dA-
SQ and dU-SQ were more soluble in polar protic solvents. Hy-
drogen bonding between deoxyribonucleoside and solvent
molecules likely facilitates this aqueous solubility.
In glycerol/water mixed solvents, negligible impact of sol-
vent polarity was evident. However, viscosity-dependent opti-
cal behaviors were observed for both deoxyribonucleoside-
modified squaraines. When the viscosity of the solution in-
creased from 1.7 cP (20% glycerol, v/v) to 209 cP (90% glycer-
ol, v/v) at 258C,[22] the absorption spectra of dU-SQ changed as
viscosity increased, with the monomer band increasing with
a concomitant decrease of the H-aggregate band. Meanwhile,
when excited at 625 nm, the fluorescence intensity of dU-SQ
was greatly enhanced (310-fold) in highly viscous solvents and
the quantum yield reached 0.16 in 90% glycerol solution
(Figure 2), a relatively high value for a squaraine dye in a polar
In some intra- and intercellular regions, high viscosity values
have been reported, which were estimated to be 100–
400 cP.[3c] We hypothesized that if the dye is in high viscosity
regions, fluorescence enhancement of dU-SQ will result with
bright emission signals collected from a dark background.
After incubation with HTC 116 (human colorectal cancer cells)
for 1 h, dU-SQ appeared to readily enter the cells, yielding
clear fluorescence images (Figure 3 and Supporting Informa-
tion, Figure S13). Hoechst was used as a nuclear stain to visual-
ize the cell nucleus for reference.
Figure 3. Images of HCT 116 cells incubated with dU-SQ (10 mm) and
Hoechst (5 mgmLÀ1): a) DIC, b) overlay of fluorescence microscopy images of
dU-SQ (red, Ex 562/40, DM 593, Em 654/40) and Hoechst (blue, Ex 377/50,
DM 409, Em 460/50), c) overlay of (a) and (b). Scale bars=10 mm.
Figure 2. a) Absorption and b) emission spectra of dU-SQ ([dU-
SQ]=1.0ꢁ10À5 m) in glycerol with varying water content. Volume ratio of
glycerol/water varied from 9:1 to 2:8. Excitation wavelength was 625 nm.
High viscosities may be generated during mitosis and in the
vicinity of microtubules (MTs).[26] Recent studies showed that
the viscosity was dependent on MT crosslinking and density.[27]
In order to confirm this, COS 7 (monkey kidney tissue cells)
were incubated with dU-SQ and Hoechst. MTs were stained
(by incubation with primary antibody anti-Vinculin mouse mAb
and subsequent staining with FITC-labeled anti-mouse goat
IgG) and different stages of mitosis were captured. During
metaphase and anaphase stages the viscosity appears higher
than telophase, as determined by the intensity (brightness) of
the fluorescence (viscosity-dependent fluorescence enhance-
ment), as shown in Figure 4 and Figure S14 in the Supporting
Information. Figure 4 presents micrographs at different stages
of mitosis, showing the DIC imaging, nuclear and MT staining,
or aqueous solvent system. dA-SQ exhibited the same behav-
ior, with a nearly 300-fold increase in fluorescence intensity in
90% glycerol solution and fluorescence quantum yield of 0.12
(Supporting Information, Figure S9).
Furthermore, at low viscosities (from 2:8 to 4:6 glycerol/
water), at which there was little change in the H-aggregates
according to the absorption spectra, the increase in fluores-
cence intensity was still very clear (Figure 2 and Supporting In-
formation, Figure S9). Thus, this ‘turn-on’ fluorescence in glyc-
erol/water mixtures can be attributed to impeding the aggre-
gation of the squaraine moiety while slowing down molecular
Chem. Eur. J. 2014, 20, 7249 – 7253
7251
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Zhang, Yuanwei
