6-Substituted quinoline-based ratiometric two-photon fluorescent probes for biological Zn2+ detection

Article abstract of DOI:10.1039/c2cc30471f

New ratiometric two-photon fluorescent probes are developed from 6-substituted quinolines for biological Zn²⁺ detection. They show large red shifts and good ratiometric responses upon Zn²⁺ binding. They also exhibit high ion selectivities and large two-photon absorption cross sections at nearly 720 nm. Because the new probes are cell-permeable, they can be used to detect intracellular zinc flux under two-photon excitation.

Full text of DOI:10.1039/c2cc30471f

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COMMUNICATION
6-Substituted quinoline-based ratiometric two-photon fluorescent probes
for biological Zn2+ detectionw
Xiangming Meng,*a Shuxin Wang,a Yiming Li,b Manzhou Zhu*a and Qingxiang Guob
Received 20th January 2012, Accepted 6th March 2012
DOI: 10.1039/c2cc30471f
New ratiometric two-photon fluorescent probes are developed
from 6-substituted quinolines for biological Zn2+ detection.
They show large red shifts and good ratiometric responses upon
Zn2+ binding. They also exhibit high ion selectivities and large
two-photon absorption cross sections at nearly 720 nm. Because
the new probes are cell-permeable, they can be used to detect
intracellular zinc flux under two-photon excitation.
of the applications.7,8 Thus the search for new ratiometric two-
photon Zn2+ fluorescent probes, in particular those with new
skeleton structures, remains an important task in the field.
In the present study, we design and synthesize new two-photon
ratiometric Zn2+ probes based on the quinoline skeleton. The
rationale for using quinoline as the basic structure is that quinoline-
based Zn2+ probes showed stable fluorescent properties, good
water solubility, and lower cell toxicity. To achieve ratiometric
detection, we consider the intramolecular charge transfer
(ICT) mechanism in our design.4,9 This mechanism involves
a charge transfer process from an electron donor to an
electron acceptor within a fluorophore. To promote ICT in
quinolines, we propose to incorporate an electron-donating
substituent at the 6-position. To improve the two-photon cross
section, we also propose to enlarge the conjugation system.
Because biphenyl does not give good conjugation due to its
intrinsic steric repulsion, we put a vinyl or alkynyl moiety into
the system ending up with probes 6-MPVQ and 6-MPQ.
As described in the ESIw, 6-MPVQ and 6-MPQ can be
easily synthesized in four steps with good yields. With the two
probes in hand, we then make their complexes with Zn2+. The
X-ray crystal structures of the two complexes (Scheme 1) show
that Zn2+ is coordinated by four nitrogen atoms. An additional
H2O- or DMF-oxygen coordinates to Zn2+ to complete the
As the second most abundant transition metal ion in the
human body, zinc is involved in many biochemical processes
including neurotransmission, enzymatic regulation and cell
apoptosis.1 However, the biological roles of Zn2+ have been
difficult to accurately understand because of its colourlessness
and magnetic silence.2 At present, fluorescent probes provide
the optimal choice to detect Zn2+ ions in biological samples
due to their high sensitivity and selectivity. Earlier Zn2+
probes are mostly designed on the basis of the technology of
single-photon fluorescence.3 Recently, as two-photon micro-
scopy (TPM) has evolved to become a widely used tool in
biology with low phototoxicity, good spatial resolution, and
increased specimen penetration,4 considerable attention has
been directed to the development of two-photon fluorescent
probes for detection of Zn2+
.
Some previous studies in this regard have led to the development
of several two-photon Zn2+ probes based on fluorescein and
coumarin.5 However, most of these two-photon Zn2+ probes
were designed to exhibit enhancement of the fluorescence intensity
at only one wavelength. This design may cause difficulty in
quantitative determination and quantitative bio-imaging due
to the background interference.6 By comparison, ratiometric
probes are better choices that can overcome this particular
limitation, because they allow quantitative detection of the
analyte by measuring the ratio of emission at two different
wavelengths.7 Up to now, very few two-photon yet ratiometric
Zn2+ probes have been successfully developed, and their
properties still need to be improved to meet the requirements
1
five-coordination geometry. Moreover, the H NMR analysis
confirms that the coordination of the probes with Zn2+ can
take place in solution (ESIw). At this point, it is interesting to
a Department of Chemistry, Anhui University, Hefei 230039,
P.R. China. E-mail: mengxm@ahu.edu.cn, zmz@ahu.edu.cn;
Fax: +86-551-5107342; Tel: +86-551-5108970
b Department of Chemistry, University of Science and Technology of
China, Hefei 230026, P.R. China
w Electronic supplementary information (ESI) available: Details of
ligand synthesis, photophysical measurements and cell imaging; NMR,
crystallographic data. See DOI: 10.1039/c2cc30471f
Scheme 1 6-MPVQ and 6-MPQ for Zn2+ detection.
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note that the conjugated system in 6-MPQ is not fully planar
as compared to that in 6-MPVQ (Scheme 1). This means that
the alkynyl connected system may enjoy a better conjugation
than the vinyl one. As shown below, such difference may cause
6-MPVQ to be advantageous over 6-MPQ for a number of
physicochemical properties.
To measure the spectroscopic properties of the two probes,
we first examine their responses to Zn2+ in methanol–water
solution (1 : 1, v/v, 50 mM HEPES, pH = 7.4). As shown in
Fig. 1 (and also ESIw), when excited at 320 nm 6-MPQ and
6-MPVQ show maximum emission at 412 and 443 nm,
respectively. The addition of Zn2+ causes the maximum
emission of 6-MPVQ to shift from 412 to 493 nm with a
significant red shift of 81 nm. Such a large red shift has been
rarely achieved for the Zn2+ probes reported to date.10 This
red shift also indicates that the nitrogen atom of the quinoline
platform is coordinated with Zn2+ causing an enhanced ICT.
Note that for 6-MPQ, the maximum emission shifts from 443
to 515 nm with a red shift of 72 nm. These large red shifts
provide a good opportunity to achieve the ratiometric detection.
As shown in Fig. 1, upon titration with Zn2+ the ratio of emission
intensity (I493 nm/I419 nm) of 6-MPVQ increases dramatically with
ca. 14-fold enhancement. A similar behavior is also observed for
6-MPQ but the enhancement is lower (7-fold).
Fig. 2 Two-photon excitation spectra of 6-MPVQ and 6-MPQ with
and without Zn2+
.
with Zn2+, the dmax values increase to 470 and 250 GM,
respectively. It is evident that 6-MPVQ shows a much better
two-photon activity than 6-MPQ, presumably because the
former corresponds to a better conjugation system. Nonetheless,
it is important to point out that the two-photon cross-section
values of both probes are significantly larger than the previously
reported ones.11
Note that the maximum ratio of fluorescence emission
intensity is obtained when the Zn2+ concentration equals that
of the probes. Job’s plot analysis also indicates that when the
Zn2+ molar fraction reaches 0.5, the maximum fluorescence
intensity is obtained indicating a 1 : 1 binding model between
the probe and Zn2+. As to the quantum yield, we find that
6-MPVQ exhibits a quantum yield enhancement of ca. 7-fold
Ion selectivity is another important property of the fluores-
cence probes. As shown in Fig. 3, high concentrations of Na+,
K+, and Ca2+ (1 mM) show negligible effects on the fluores-
cence of 6-MPVQ (also for 6-MPQ as shown in the ESIw).
Besides, our tests show that both the probes are pH-insensitive
in the biologically relevant pH range (ESIw). Moreover, other
metal ions including Co2+, Cr3+, Cu2+, Fe2+, Mg2+, Mn2+
,
(from Ffree = 0.036 to FZn2+ = 0.266) upon binding to Zn2+
.
and Ni2+ do not interfere with the probe. Nonetheless, Cd2+
exhibits some enhancement of the fluorescence, which is a
phenomenon observed for many previously developed Zn2+
probes.12 Fortunately, the interference of Cd2+ is negligible in
living cells. As a result, we believe that the present probes
should have good selectivity for Zn2+ in the biological studies.
To investigate the use of 6-MPVQ and 6-MPQ in vivo under
two photon excitation, HeLa cells are cultured and stained
with the two probes within 30 min respectively. The cells are
then washed once and bathed in Dulbecco’s modified Eagle
medium (DMEM) containing no fetal calf serum (FCS) prior
By comparison, the quantum yield of 6-MPQ increases by
ca. 5-fold. The apparent dissociation constants (Kd) of 6-MPVQ
and 6-MPQ with Zn2+ are measured to be 0.45 and 0.58 nM
respectively. Thus, both the probes can detect free Zn2+ in the
nanomolar range, which affords sufficient sensitivity for Zn2+ in
living cells.
The two-photon cross sections of the two probes and their
Zn2+ complexes are determined by using the two-photon induced
fluorescence measurement technique. As shown in Fig. 2,
6-MPVQ and 6-MPQ show the dmax values of 335 GM at
710 nm and 135 GM at 725 nm, respectively. Upon saturating
Fig. 1 (a) Emission spectra of 6-MPVQ (25 mM) with the excitation
at 356 nm upon titration with Zn2+ (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,
0.8, 0.9, 1.0, 1.2, 1.4 and 1.6 equiv.) in the methanol–water solutions
(1 : 1, v/v, 50 mM HEPES buffer, pH = 7.4); (b) ratiometric calibration
curve between 419 and 493 nm (I493 nm/I419 nm) as a function of Zn2+
concentration.
Fig. 3 Fluorescence ratio (493 nm/412 nm) of 6-MPVQ upon addition
of various metal ions. Experimental conditions: 25 mM 6-MPVQ,
1.0 mM for Na+, K+, and Ca2+, and 25 mM for Co2+, Cr3+, Cu+
Cu2+, Fe2+, Mg2+, Mn2+, Ni2+, Zn2+ and Cd2+, lex = 320 nm.
,
c
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We acknowledge financial support by NSFC (21102002,
21072001 and 20932006), Anhui Province Natural Science
Foundation (090416231), Natural Science Foundation of
Education Department of Anhui Province (KJ2010A028), and
211 Project of Anhui University for supporting the research.
Notes and references
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Fig. 4 (a) Two-photon image of HeLa cells incubated with 15 mM
6-MPVQ after 30 min of incubation, washed with PBS buffer. lex
=
800 nm (emission wavelength from 390 to 465 nm). (b) Emission
wavelength from 500 to 550 nm. (c) Bright-field image of HeLa cells.
(d) The overlay of (a), (b) and (c). (e) Two-photon image following a
30 min treatment with Zn2+/pyrithione (30 mM, 1 : 1 ratio). Emission
wavelength from 390 to 465 nm. (f) Emission wavelength from 500
to 550 nm. (g) Bright-field image of HeLa cells. (h) The overlay of (e),
(f) and (g).
to imaging and/or Zn2+ addition. TPM images are then obtained
by exciting the probes with a mode-locked titanium–sapphire
laser source set at wavelength 800 nm. According to the
fluorescent properties of the probes, the optical windows at
390–465 and 500–530 nm are chosen for confocal imaging of
6-MPVQ. As shown in Fig. 4, before addition of Zn2+ the
optical window at 390–465 nm shows moderate fluorescence,
whereas the optical window at 500–530 nm exhibits weak
fluorescence. This experiment indicates that the new probes
are cell permeable. After Zn2+ is added to the cells, the
fluorescence intensity of the optical window at 500–530 nm
increases dramatically, whereas that at 390–465 nm significantly
decreases. Similar behaviors are also observed with 6-MPQ
under TPM excitation (ESIw). Thus the ratiometric fluorescence
images generated from the above optical windows demonstrate
that the new probes can reveal the variation of the intracellular
zinc flux in vivo system under two-photon excitation.13 Moreover,
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(MTT) assay demonstrates that the probes show essentially
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In summary, we have developed two 6-substituted quinoline-
based ratiometric two-photon zinc probes (6-MPVQ and 6-MPQ)
and investigated their applications in vitro and in vivo. Among
the two probes, 6-MPVQ shows a large red shift upon Zn2+
binding with 14-fold emission enhancement and a significant
increase of the two-photon cross section. Moreover, 6-MPVQ
exhibits high ion selectivity and sensitivity for Zn2+ in a
neutral aqueous solution. The in vivo two-photon microscopy
imaging experiments show that the new probes are cell permeable
and can be used for imaging Zn2+ in living cells in a ratiometric
fashion. The good two-photon properties of the 6-substituted
quinolines also hint the potential of their applications to the
design of probes for other metal ions.
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