A novel NIR fluorescent turn-on sensor for the detection of pyrophosphate anion in complete water system

Article abstract of DOI:10.1039/c2cc16902a

A novel fluorescent sensor DCAA-Cu²⁺ was developed, showing turn-on fluorescence in NIR region with high selectivity to pyrophosphate anion in 100% aqueous solution.

Full text of DOI:10.1039/c2cc16902a

View Article Online / Journal Homepage / Table of Contents for this issue
Dynamic Article Links
ChemComm
Cite this: Chem. Commun., 2012, 48, 1784–1786
www.rsc.org/chemcomm
COMMUNICATION
A novel NIR fluorescent turn-on sensor for the detection of
pyrophosphate anion in complete water systemw
Weihong Zhu,* Xiaomei Huang, Zhiqian Guo, Xumeng Wu, Huihui Yu and He Tian
Received 8th November 2011, Accepted 6th December 2011
DOI: 10.1039/c2cc16902a
A novel fluorescent sensor DCAA-Cu2+ was developed, showing
turn-on fluorescence in NIR region with high selectivity to
pyrophosphate anion in 100% aqueous solution.
The selective recognition and sensing of biologically important
anions has attracted great attention for both chemists and
biologists.1,2 Near-infrared (NIR) dyes with emission wave-
lengths in the region of 650–900 nm are preferable to bio-
imaging with several advantages, such as penetrating deeper
into the tissue with smaller scattering, lower damage of living-cells,
and less auto-fluorescence with much lower background signal.3
As the bioproduct of adenosine triphosphate (ATP) hydrolysis
Scheme 1 The synthetic route of DCAA: (i) BrCH2COOEt,
and enzymatic reactions, pyrophosphate anion (PPi) is becoming
a biologically important target.4 Generally, metal complexes can
bind anions more efficiently than water, therefore the utilization
of a metal ion complex as a binding site for PPi has become the
most popular approach.5 However, the development in fluores-
cent sensors for PPi under real aqueous physiological conditions
is still challenge due to the strong hydration effects and various
complexation geometries.6 To date, there are a few chemical
sensors that detect PPi in an aqueous solution,7 however, the
emission spectra are in short wavelength and cannot fall in the
NIR region. Accordingly, it is of great significance to develop
NIR sensors that can detect PPi selectively and sensitively in a
100% aqueous physiological system.
K2HPO4/CH3CN, 75% yield; (ii) POCl3, DMF, 60% yield; (iii) 2-(2-
methyl-4H-chromen-4-ylidene) malononitrile, 48% yield; (iv) LiOH,
MeOH/THF, 48% yield.
knowledge, DCAA–Cu2+ is the first NIR fluorescent sensor
for PPi with the emission wavelengths beyond 650 nm, especially
in complete water solution.
As shown in Scheme 1, the sensor precursor DCEA was
first obtained by a Knoevenagel reaction of 2-(2-methyl-4H-
chromen-4-ylidene)malononitrile and diethyl 2,20-((4-formyl-
phenyl)azanediyl)diacetate (2), which was synthesized from
diethyl 2,20-(phenylazanediyl)diacetate (1) via Vilsmeier
formylation in 60% yield.9 Then, the target lithium iminodi-
acetate DCAA was obtained from the hydrolysis of DCEA
under basic conditions using a modified protocol.10 The
chemical structures were fully characterized by 1H NMR,
13C NMR and HRMS, as shown in the electronic supple-
mentary information (ESIw). The characteristic coupling
Bearing this in mind, herein we present a novel colorimetric
and fluorescent turn-on PPi sensor DCAA–Cu2+, which
employs dicyanomethylene-4H-chromene as the fluorophore
and iminodiacetic acid group as the receptor. Since a-amino
acid is a good chelating ligand for Cu2+,8 the incorporation of
a lithium iminodiacetate group in DCAA (Scheme 1) can play
a double role, that is, as the coordinate receptor and to
improve water solubility. More importantly, the system can
preferably extend the conjugation system with a red shift in
emission band to 675 nm, thus making the determining
wavelength fall in the desired NIR region. To the best of our
1
constant (J = 16.0 Hz) of the protons in H NMR of DCAA
is indicative of the predominate trans-isomer (Scheme 1). In
electronic spray source HRMS, the high resolution mass peak
found at 426.1089 is consistent with the calculated data for
[M ꢀ 2Li + H]ꢀ (426.1090).
Due to the incorporation of a highly hydrophilic group with
the lithium iminodiacetate group, DCAA is completely soluble
in water. Hence, the fluorescent behavior of DCAA toward
various metal ions were first checked in a completely aqueous
system buffered with 3-(N-morpholino) propanesulfonic acid
(MOPS, 10 mM, pH = 7.0). As shown in Fig. 1A, there is no
obvious change in the fluorescent emission in the presence of
Shanghai Key Laboratory of Functional Materials Chemistry,
Key Laboratory for Advanced Materials and Institute of Fine
Chemicals, East China University of Science & Technology,
Shanghai 200237, P. R. China. E-mail: whzhu@ecust.edu.cn;
Fax: (+86) 21-6425-2758
w Electronic supplementary information (ESI) available: Synthesis
and full characterization of DCAA, Figs S1–S12. See DOI: 10.1039/
c2cc16902a
K+, Na+, Mg2+, Ca2+, Zn2+, Ag+, Cd2+, Hg2+, Co2+
,
Mn2+, Ni2+ and Sn2+, but only adding Cu2+ causes a
c
1784 Chem. Commun., 2012, 48, 1784–1786
This journal is The Royal Society of Chemistry 2012

View Article Online
Based on the quenching character of Cu2+ to DCAA, the
complex of DCAA with Cu2+ was prepared in situ by mixing
DCAA with Cu(ClO4)2 at a 1 : 5 ratio in aqueous solution,3a
then the selectivity of DCAA–Cu2+ toward different anions
was determined. As shown in Fig. 1B, various anions (1, Fꢀ;
2ꢀ
2ꢀ
2, Clꢀ; 3, Brꢀ; 4, Iꢀ; 5, CH3COOꢀ; 6, H2PO4ꢀ; 7, HPO4
8, PO43ꢀ; 9, P2O74ꢀ; 10, SO42ꢀ; 11, NO3ꢀ; 12, CO3
;
;
13, HSO3ꢀ; 14, NO2ꢀ; 15, HCO3ꢀ) were added into the solution,
but only PPi responded obviously to the sensor; other phosphate
2ꢀ
anions, such as H2PO4ꢀ, HPO4 and PO43ꢀ, showed a minor
disturbance. Apparently, DCAA–Cu2+ can function as a
fluorescent turn-on PPi sensor from other investigated anions,
including phosphate anions. However, the system cannot avoid
the possible disturbance of peptides and amino acids, such as
glycine and histidine at high concentration (Fig. S4w).12
PPi titration was then conducted in an aqueous system
buffered with MOPS (10 mM, pH = 7.0). When PPi was
gradually added to the solution of DCAA–Cu2+, a new red-
shifted peak at 503 nm appeared and increased gradually with
an isosbestic point at 450 nm (Fig. 2A). The associated color
change from pale brown to red can be easily distinguished by
the naked eye (Fig. 3). Meanwhile, the fluorescence intensity in
the NIR region of 675 nm is obviously enhanced (Fig. 2B) and
gradually stabilized upon the addition of 15 equiv. of PPi.
Choosing the well-known laser dye DCM as a standard
(FDCM = 0.44),13 the fluorescence quantum efficiency (F) of
DCAA is calculated to be 0.79 and that of DCAA–Cu2+ after
the addition of PPi is 0.48. Here, the fluorescence can’t be
recovered to the original intensity of DCAA, which indirectly
indicates that PPi does not form a strong enough complex to
remove Cu2+ from DCAA–Cu2+ (Fig. S5w). It is most likely
that one DCAA is displaced by PPi, partly restoring the
emission of the ligand. Similarly, the detection limit of
DCAA–Cu2+ toward PPi was evaluated to be 2.02 mM using
3 mM DCAA–Cu2+, which is superior or comparable to most
of the reported PPi sensors (Fig. S6w).14
Fig. 1 (A) The fluorescence change of DCAA (10 mM) to various
metal ions at 50 mM concentration in an aqueous solution buffered
with MOPS (10 mM, pH = 7.0) excited at 500 nm. Note: the
corresponding perchlorates were utilized as the source of metal
cations. (B) The fluorescent response of DCAA–Cu2+ (10 mM) to
various anions at 150 mM concentration in an aqueous solution
buffered with MOPS (10 mM, pH = 7.0) excited at the isosbestꢀic
point of 450 nm: 1, Fꢀ; 2, Clꢀ; 3, Brꢀ; 4, Iꢀ; 5, CH3COOꢀ; 6, H2PO4
;
7, HPO42ꢀ; 8, PO43ꢀ; 9, P2O74ꢀꢀ; 10, SO42ꢀ; 11, NO3ꢀ; 12, CO32ꢀ; 13,
HSO3ꢀ; 14, NO2ꢀ; 15, HCO3
.
significant decrease in fluorescence, indicating that DCAA can
distinguish Cu2+ from other metal ions in neutral aqueous
buffer solution. Consequently, the copper titration experiment
was further investigated. As shown in Fig. S1w, the fluorescent
intensity decreased with an increase in Cu2+ concentration
and complete fluorescence quenching was observed when
5.0 equiv. of Cu2+ was added. Moreover, the Job plots with
both absorption and fluorescence titrations exhibited
a
Finally, we applied the NIR sensor of DCAA–Cu2+ to KB
cells (human nasopharyngeal epidermal carcinoma cell) with
the use of a confocal fluorescence microscopy to examine the
potential application in bioimaging. Bright-field measurements
confirmed that the cells after treatment with DCAA–Cu2+ and
PPi were viable throughout the imaging experiments. Incubation
of KB cells with 10 mM of DCAA–Cu2+ for 0.5 h at 37 1C gave
nearly no intracellular fluorescence (Fig. 4A–C). After the cells
were subsequently supplemented with PPi (30 mM) for 30 min
at 37 1C, a remarkable increase of intracellular fluorescence
maximum at about 0.33 mol fraction (Fig. S1w), indicating
that DCAA forms a 2 : 1 complex with Cu2+, which is con-
sistent with the binding mode of corresponding complexes.11
Also, the mass peak for DCAA–Cu2+ at m/z 915.1472 corres-
ponding to [C48H32N6O10Cu]ꢀ (= [2DCAA + Cu2+ + 2H]ꢀ
calculated as 915.1476) gives strong evidence that DCAA
forms a 2 : 1 complex with Cu2+ (Fig. S1Cw). The binding
constant of DCAA with Cu2+ was determined to be 1.1 ꢁ
10ꢀ6 Mꢀ1 based on fluorescence titrations.
Interestingly, when adding 1.0 equiv. of EDTA solution, the
fluorescence intensity was fully restored (Fig. S2w), which can
be explained by the difference in association constants between
EDTA–Cu2+ and DCAA–Cu2+. As a consequence, the
formation of metal complex of DCAA–Cu2+ is responsible
for the fluorescence quenching. The sensitivity to Cu2+ was
also calculated on the basis of the linear relationship between
the emission intensity at 675 nm and the Cu2+ concentration,
indicating that DCAA has a detection limit of 1.26 mM for
Cu2+ in aqueous solution (Fig. S3w), which is much lower
than the typical concentration of blood copper (11.8–23.6 mM)
in normal individuals and the limit of copper in drinking water
(B20 mM) set by the U. S. Environmental Protection Agency.
Fig. 2 Spectra of DCAA–Cu2+ (10 mM) in an aqueous solution
buffered with MOPS (10 mM, pH = 7.0) upon titration with
0–15 equiv. PPi: (A) absorption change; (B) emission change excited
at the isosbestic point of 450 nm.
c
This journal is The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 1784–1786 1785

View Article Online
Notes and references
1 (a) E. L. Que, D. W. Domaille and C. J. Chang, Chem. Rev., 2008,
108, 1517; (b) R. W. Sinkeldam, N. J. Greco and Y. Tor, Chem.
Rev., 2010, 110, 2579; (c) J. L. Liu, Y. Liu, Q. Liu, C. Y. Li,
L. N. Sun and F. Y. Li, J. Am. Chem. Soc., 2011, 133, 15276.
2 (a) J. F. Callan, A. P. de Silva and D. C. Magri, Tetrahedron, 2005,
61, 8551; (b) R. M. Duke, E. B. Veale, F. M. Pfeffer, P. E. Kruger
and T. Gunnlaugsson, Chem. Soc. Rev., 2010, 39, 3936.
3 (a) X. Chen, S. Nam, G. Kim, N. Song, Y. Jeong, I. Shin,
S. K. Kim, J. Kim, S. Park and J. Yoon, Chem. Commun., 2010,
46, 8953; (b) M. Zhu, M. Yuan, X. Liu, J. Xu, J. Lv, C. Huang,
H. Liu, Y. Li, S. Wang and D. Zhu, Org. Lett., 2008, 10, 481;
(c) S. Sreejith, K. P. Divya and A. Ajayaghosh, Angew. Chem., Int.
Ed., 2008, 47, 7883; (d) C. Y. Li, M. X. Yu, Y. Sun, Y. Q. Wu,
C. H. Huang and F. Y. Li, J. Am. Chem. Soc., 2011, 133, 11231;
(e) X. J. Peng, Z. G. Yang, J. Y. Wang, J. L. Fan, Y. X. He,
F. L. Song, B. S. Wang, S. G. Sun, J. L. Qu, J. Qi and M. Yang,
J. Am. Chem. Soc., 2011, 133, 6626.
Fig. 3 Photographic images observed from DCAA–Cu2+ with the
addition of PPi ([DCAA–Cu2+] = 10ꢀ4 M, [PPi] = 5 ꢁ 10ꢀ4 M):
(A) color change; (B) fluorescent emission change irradiated at 365 nm
by a portable fluorescent lamp.
4 (a) H. N. Lee, Z. Xu, S. K. Kim, K. M. K. Swamy, Y. Kim,
S. J. Kim and J. Yoon, J. Am. Chem. Soc., 2007, 129, 3828;
(b) J. K. Heinonen, Biological Role of Inorganic Pyrophosphate,
Kluwer Academic Publishers, Norwell, 2001.
5 (a) R. Villamil-Ramos and A. K. Yatsimirsky, Chem. Commun.,
2011, 47, 2694; (b) M. J. Kim, K. M. K. Swamy, K. M. Lee,
A. R. Jagdale, Y. Kim, S. Kim, K. H. Yoo and J. Yoon, Chem.
Commun., 2009, 7215; (c) H. Wu, C. He, Z. Lin, Y. Liu and
C. Duan, Inorg. Chem., 2009, 48, 408; (d) M. Kruppa and
B. Konig, Chem. Rev., 2006, 106, 3520; F. Schmidt,
¨
¨
S. Stadlbauer and B. Konig, Dalton Trans., 2010, 39, 7250;
(e) D. Ramaiah, P. P. Neelakandan, A. K. Nair and
R. R. Avirah, Chem. Soc. Rev., 2010, 39, 4158; (f) X. M. Huang,
Z. Q. Guo, W. H. Zhu, Y. S. Xie and H. Tian, Chem. Commun.,
2008, 5143.
6 (a) J. Wen, Z. Geng, Y. Yin, Z. Zhang and Z. Wang, Dalton
Trans., 2011, 40, 1984; (b) W. Chen, Y. Xing and Y. Pang, Org.
Lett., 2011, 13, 1362; (c) S. K. Kim, D. H. Lee, J. I. Hong and
J. Yoon, Acc. Chem. Res., 2009, 42, 23.
7 For water soluble PPi sensors: (a) T. Cheng, T. Wang, W. Zhu,
X. Chen, Y. Yang, Y. Xu and X. Qian, Org. Lett., 2011, 13, 3656;
(b) H. J. Kim, J. H. Lee and J. I. Hong, Tetrahedron Lett., 2011,
52, 4944; (c) C. R. Lohani, J. M. Kim, S. Y. Chung, J. Yoon and
K. Lee, Analyst, 2010, 135, 2079; (d) X. Zhao and K. S. Schanze,
Chem. Commun., 2010, 46, 6075; (e) B. Gruber, S. Stadlbauer,
Fig. 4 Confocal fluorescence images in KB cells: Top, (A–C) cells
incubated with DCAA–Cu2+ (10 mM) for 0.5 h. Bottom, (D–F) cells
incubated with DCAA–Cu2+ (10 mM) for 0.5 h, then K4P2O7 (30 mM)
for 0.5 h. Emission was collected at 630–730 nm upon excitation at
405 nm. Bright field (A and D), fluorescence (B and E) and overlap
field (C and F).
was observed (Fig. 4D–F). The overlay of fluorescence and
bright-field images reveals that the fluorescence signals are
localized in the perinuclear area of the cytosol, indicating a
subcellular distribution of PPi anion and good cell membrane
permeability of DCAA–Cu2+. Moreover, from the bioimaging
of cells (Fig. 4A–C), we found that DCAA–Cu2+ did not show
fluorescent enhancement, suggesting that the disturbance of
peptide and amino acids in the cell environment can be
neglected. This cell experiment demonstrated the potential
application of DCAA–Cu2+ for the imaging of PPi in
living cells.
In conclusion, a novel fluorescent sensor DCAA–Cu2+ was
designed on the basis of a dicyanomethylene-4H-chromene
framework with several merits: (i) detecting PPi anion selec-
tively and sensitively in a pure aqueous physiological system;
(ii) being capable of the maximum signal-to-noise ratio with
the preferable ‘off–on’ sensing mode; and (iii) falling NIR
determining wavelength to get good transmission and low
autofluorescence in biological samples.
A. Spath, S. Weiss, M. Kalinina and B. Konig, Angew. Chem., Int.
¨
Ed., 2010, 49, 7125.
¨
8 (a) E. L. Que and C. J. Chang, J. Am. Chem. Soc., 2006,
128, 15942; (b) K. Varazo, F. Xie, D. Gulledge and Q. Wang,
Tetrahedron Lett., 2008, 49, 5293; (c) N. Kaur and S. Kumar,
Tetrahedron Lett., 2006, 47, 4109; (d) M. Yu, M. Shi, Z. Chen,
F. Li, X. Li, Y. Gao, J. Xu, H. Yang, Z. Zhou, T. Yi and
C. Huang, Chem.–Eur. J., 2008, 14, 6892.
9 X. Meng, M. Z. Zhu, L. Liu and Q. X. Guo, Tetrahedron Lett.,
2006, 47, 1559.
10 Q. W. He, E. W. Miller, A. P. Wong and C. J. Chang, J. Am.
Chem. Soc., 2006, 128, 9316.
11 (a) K. Varazo, F. Xie, D. Gulledge and Q. Wang, Tetrahedron Lett.,
2008, 49, 5293; (b) M. Choi, M. Kim, K. D. Lee, K. N. Han,
I. A. Yoon, H. J. Chung and J. Yoon, Org. Lett., 2001, 3, 3455;
(c) L. X. Wu, Y. R. Dai and G. Marriott, Org. Lett., 2011, 13, 2018.
12 (a) M. Kruppa and B. Konig, Chem. Rev., 2006, 106, 3520;
¨
(b) S. Sun, M. A. Fazal, B. C. Roy and S. Mallik, Org. Lett.,
2000, 2, 911.
13 J. M. Drake, M. L. Lesiecki and D. M. Camaioni, Chem. Phys.
Lett., 1985, 113, 530.
14 (a) D. H. Lee, S. Y. Kim and J. I. Hong, Angew. Chem., Int. Ed.,
2004, 43, 4777; (b) F. Zapata, A. Caballero, A. Espinosa,
A. Tarraga and P. Molina, J. Org. Chem., 2008, 73, 4034;
´
We are grateful for support from NSFC/China, the Oriental
Scholarship, SRFDP 200802510011, the Fundamental
Research Funds for the Central Universities (WK1013002)
and STCSM (10dz2220500).
(c) T. M. Fu, C. Y. Wu, C. C. Cheng, C. R. Yang and
Y. P. Yen, Sens. Actuators, B, 2010, 146, 171.
c
1786 Chem. Commun., 2012, 48, 1784–1786
This journal is The Royal Society of Chemistry 2012