Bay functionalized perylenediimide as a deaggregation based intracellular fluorescent probe for perchlorate

Article abstract of DOI:10.1039/c4cc06765g

The aggregates of perylenediimide based chemosensor (PDI 1) undergo de-aggregation induced fluorescence quenching selectively with ClO₄^-ions both in the solution and in the solid phase and can detect ClO₄^-ions in drinking water and fireworks. PDI 1 is permeable to C6 glioma cells, and ClO₄^-can be detected using confocal microscopy.

Full text of DOI:10.1039/c4cc06765g

ChemComm
View Article Online
View Journal | View Issue
COMMUNICATION
Bay functionalized perylenediimide as a
deaggregation based intracellular fluorescent
probe for perchlorate†
Cite this: Chem. Commun., 2014,
0, 13994
5
Received 27th August 2014,
Accepted 12th September 2014
a
a
b
a
Prabhpreet Singh,* Lalit Singh Mittal, Vanita Vanita, Rahul Kumar,
c
b
a
Gaurav Bhargava, Amandeep Walia and Subodh Kumar
DOI: 10.1039/c4cc06765g
www.rsc.org/chemcomm
The aggregates of perylenediimide based chemosensor (PDI 1) undergo due to high water solubility, may be transported from soil to
4À
4À
de-aggregation induced fluorescence quenching selectively with ClO
ions both in the solution and in the solid phase and can detect ClO
the surface or ground water and then become potential health
concern because perchlorates can impair proper functioning of
1
4
ions in drinking water and fireworks. PDI 1 is permeable to C6 glioma the thyroid gland.
4À
4À
cells, and ClO can be detected using confocal microscopy.
Current strategies for ClO detection require long proces-
sing times, expensive instruments, and in many cases the
1
5,16
For the last few years, research has focused on developing chromo- techniques have poor selectivity and high detection limits.
fluorescent sensors as a safeguard for global ecosystems, and for Recently, we have shown that N-aryl benzimidazolium moieties
monitoring of toxic anions and carcinogenic metal ions that linked through an appropriate spacer can be used for selective
1
,2
À
17
impose serious human and environmental health hazards.
Perylenediimides (PDI) are intensively coloured dyes with high
molar extinction coefficients, fluorescence quantum yields, unique undergoes fluorescence quenching with only ClO
detection of ClO4 ions.
Herein we report a PDI based chemosensor, PDI 1, which
À
4
ions in
light harvesting and redox properties and exceptional thermal, HEPES buffer (10% DMSO) (v/v) (pH 7.4) with a minimum limit
3
chemical and photochemical stability. Although literature reports of detection of 60 nM. PDI 1 was also evaluated for the detection
À
have considered PDIs as promising materials for various opto- of ClO
4
in drinking water and fireworks. In solid phase, TLC
4
,5
À2
À
electronic devices, we have witnessed slow growth in consider- strips doped with PDI 1 can detect 0.8 ng cm of ClO
4
under
ing PDIs as chromo-fluorescent probes for sensing applications. UV-illumination. The cell uptake studies of PDI 1 with live rat
Probably their poor solubility in aqueous media is one of the C6 glioma cells show permeability of PDI 1 into the cytoplasm
À
most detrimental factors for developing molecular recognition followed by quenching of fluorescence with ClO4 ions in a
receptors. So far, only a few reports on PDI based fluorescent concentration-dependent manner.
6
–10
6,11,12
probes for cations
and anions
have been reported and
PDI 1 was synthesized by nucleophilic substitution of PDI 3
with hydroxyphenylbenzimidazole followed by heating of PDI 2
À
none of these were for perchlorate ions (ClO
4
).
Perchlorates find extensive use as oxidants in solid rocket and 1-bromobutane mixture in DMF:CH
fuel, fireworks and pyrotechnic devices, flares, electrolyte in (See ESI† for synthesis details and structural characterization)
lithium batteries, gas generator, pharmaceutical for hyper- Before examining the solution properties of PDI 1 towards
3
CN at 100 1C (Scheme 1).
thyroid and chemistry reagents. In nature, perchlorates may be different anions, the aggregation behaviour of PDI 1 in DMSO
formed by photochemical transformation reactions involving and a binary mixture of DMSO:H O (v/v) was investigated. The
2
1
3
chlorine precursors and volcanic eruptions. Perchlorates, concentration-dependent UV-Vis and emission experiments
À3
À6
(
1
1
1 Â 10 M to 1 Â 10 M) show a minor propensity of PDI
for aggregation in DMSO with an A0–0/A0–1 ratio greater than
a
Department of Chemistry, UGC Centre for Advanced Studies,
Guru Nanak Dev University, Amritsar 143 005, India.
E-mail: prabhpreet.chem@gndu.ac.in; Tel: +91-84271-01534
Department of Human Genetics, Guru Nanak Dev University, Amritsar 143 005,
India
.4 (Fig. 1a). The solvent-dependent UV-Vis and emission
experiments with different DMSO–H O compositions (starting
2
from pure DMSO and increasing the volume fraction of H O) at
a fixed concentration of 10 mM show that at low volume fraction
b
c
2
Department of Applied Sciences, Punjab Technical University, Kapurthala-144601,
Punjab, India
of water [DMSO : H O 8 : 2 (v/v)], the absorption spectrum
2
exhibits well-resolved vibronic band for A0–0 at 526 nm, whereas
the absorption band for A0–1 at 500 nm was poorly resolved. The
value of the ratio A0–0/A0–1 drastically decreased from 1.4 to 0.7 as
†
Electronic supplementary information (ESI) available: Experimental details,
additional photophysical, spectroscopic and microscopic characterization of
À
PDI 1 and PDI 1CClO
4
complex. See DOI: 10.1039/c4cc06765g
13994 | Chem. Commun., 2014, 50, 13994--13997
This journal is ©The Royal Society of Chemistry 2014

View Article Online
Communication
ChemComm
2 3
Scheme 1 Synthesis of PDI 1; reagents and conditions: (a) NMP, K CO , (7),
8
3
0 1C, 8 h; (b) 1-bromobutane, DMF:CH CN, 100 1C, 48 h.
Fig. 2 SEM micrograph (a and b) and DLS bar graph (c and d) of 5 mM (90%
aqueous DMSO solution) of PDI 1 alone and after addition of perchlorate
ions (40 mM).
Fig. 1 (a) Concentration-dependent UV-Vis absorption spectra of PDI 1
À6
À4
(
10 to 10 M) in pure DMSO; (b) solvent-dependent UV-Vis absorption
changes of PDI 1 after incremental addition of 10 vol% of H O in DMSO at a
concentration of 10 mM. Inset: correlation plot of DG1 values against vf
volume fraction) of H O.
4À
emission intensity upon addition of ClO (Fig. 3a), whereas
2
the addition of all other anions (100 mM) causes insignificant
change in its emission spectrum.
(
2
The fluorescence intensity of PDI 1 at 577 nm gradually
À
decreases as the concentration of ClO4 is increased from
the percentage of H O increased to 90% (Fig. 1b) (see Fig. S1, 0–70 mM and then it achieves a plateau. The Stern–Volmer plot
2
ESI†). Fig. 1b also shows the appearance of new bathochromically follows the linear relation up to the concentration range of
shifted absorption band at 580 nm with isosbestic point at 0–30 mM (Fig. 3b, inset). The Stern–Volmer constant (K ) is
sv
5
À1
5
53 nm, thus showing a transition from the molecularly dissolved 1.65 Â 10 M and the quenching rate constant (K ) of PDI 1
q
18
À
13
À1 À1
state to the H-type excitonically coupled aggregates. The correla- for ClO
tion plot of DG1 versus H
is 3.8 Â 10
M
s . The minimum limit of
4
1
9
À
2
O content in ratios from 0 to 0.9 shows a detection for ClO is 60 nM. The Job’s plot shows inflection
4
linear relationship (Fig. 1b inset). The fluorescence intensity of at the mole fraction of 0.5 with a symmetrical peak shape and
À
PDI 1 remained unaffected between pH 8.0 and 4.5 (see Fig. S2, suggests the formation of a PDI 1 : ClO4 (1 : 1) complex. The
À
ESI†) and allowed for the selection of HEPES buffer–10% DMSO association constant (K) of PDI 1 with ClO4 as determined by
5
À1
(
v/v) (pH 7.4) solutions for examining its anion sensing ability, the Benesi–Hildebrand plot is 0.56 Â 10
M . The HRMS
where PDI 1 exists in an aggregated state.
The aggregation of PDI 1 in DMSO–H
spectrum of 1 : 1 solution of PDI 1 and NaClO shows a parent
O (1 : 9) is further ion peak at m/z 1183.4296 (theor. 1183.44) and confirms the
4
2
confirmed by SEM and DLS experiments. The SEM images of formation of a 1 : 1 complex (see Fig. S4, ESI†). We also
À
thin films of PDI 1 reveal the formation of aggregates with an observed that the fluorescence quenching efficiency of ClO4
average diameter of 0.8–1.5 mm and are consistent with the
1–1.2 mm size of aggregates observed in DLS experiments
(Fig. 2a and c). In wide angle X-ray diffraction (WXRD) of PDI 1,
the presence of a broad peak around 2y E 18.251 (d = 4.77 Å)
points to loose packing of chains at the 1,7-positions, and peaks
around 2y = 25.641 (d = 3.47 Å) and 2y = 26.621 (d = 3.34 Å) indicate
the p–p stacking between adjacent perylene rings.
The UV-Vis spectrum of PDI 1 (10 mM), recorded in HEPES
buffer–10% DMSO (v/v), shows lmax at 509 nm, whereas the
emission spectrum of 1 (10 mM), on excitation at 500 nm,
displays emission band at lmax 577 nm. The UV-Vis spectrum
of PDI 1 shows insignificant change in the absorption spectrum
Fig. 3 (a) Fluorescence intensity changes of PDI 1 (10 mM) in the presence of
various anions recorded in HEPES buffer (0.01 M, pH 7.4)–10% DMSO (v/v);
À
À
À
À
À
À
upon addition of anions viz. F , Cl , I , CN , OH , AcO ,
À
(
b) fluorescence spectra of PDI 1 (10 mM) upon addition of the ClO
4
ions;
À
2À
À
2À
2À
ClO
4
, CO
3
, H
2
PO
4
, NO
3
, SO
4
(see Fig. S3, ESI†). The
excitation at 500 nm. Inset: Stern–Volmer plot at 577 nm corresponds to
emission spectrum of PDI 1 shows >90% quenching of the fluorescence titration.
This journal is ©The Royal Society of Chemistry 2014
Chem. Commun., 2014, 50, 13994--13997 | 13995

View Article Online
ChemComm
Communication
was not significantly affected in the presence of other probable found to be 3 mM from the standard calibration curve, leading
À4
interfering anions (see Fig. S5, ESI†).
The DLS and SEM experiments show the de-aggregation of solution (see Fig. S9, ESI†).
PDI 1 aggregates upon addition of ClO4 ions. The size of the
to the total concentration of 3.7 Â 10 M for firework sample
À
À
Contact mode analysis of ClO4 ions via TLC strips coated with
aggregates is reduced from 1–1.2 mm size to mixture of 200 nm PDI 1 was also studied under illumination of 365 nm UV light. We
À7
À
4
causes naked
and 600–700 nm size aggregates as observed by both the observed that the addition of 6 mL of 10 M ClO
techniques (Fig. 2). The SEM coupled energy dispersive X-ray eye observable quenching of the fluorescence intensity of PDI 1.
spectroscopy (EDX) confirms the presence of N and Cl on the However, complete quenching of fluorescence intensity was
À
À6
À
surface of the spherical structure PDI 1 + ClO
4
complex (see observed with 6 mL of 10 M ClO
4
. PDI 1 shows no quenching
À
Fig. S6, ESI†). The DLS experiments of the PDI 1 + ClO
4
(1 : 4) of fluorescence with only water (as control). Addition of higher
complex also show the decrease in size of the aggregates from concentrations caused reddish colour area developed on the TLC
À
1
–1.2 mm to 200 and 600–700 nm (Fig. 2b and d).
Significantly, the fluorescence titration of PDI 1 with ClO4
strip itself (Fig. 5) and indicates the presence of excess ClO
.
4
À
À2
À
Therefore, the minimum 0.8 ng cm of ClO4 is detectable.
4À
in HEPES buffer–DMSO (1 : 1; v/v) (pH 7.4), i.e. in the non-
aggregate state of PDI 1, shows poor sensitivity towards ClO
To test the biological application of PDI 1 to detect ClO in
the live cells, glial cells of the rat brain (C6 glioma cells) have
À
4
(see Fig. S7, ESI†). Therefore, the results of fluorescence studies been used for cell imaging studies (Fig. 6). Brightfield image
are consistent with SEM and DLS experiments, showing that shows that the C6 glioma cells were healthy and viable through-
À
ClO
4
mediated de-aggregation of PDI 1 is responsible for its out the experiment [Fig. 6(a)]. C6 glioma cells, themselves
À
fluorescence quenching.
(without PDI 1) and after incubation with ClO4 (40 mM) did
not exhibit any fluorescence [Fig. 6(b) and (c)].
À
The optimization of the 1 : 1 geometry of PDI 1 and ClO4
shows that the two C –H moieties of benzimidazolium groups
However, C6 glioma cells on incubation with PDI 1 (10 mM)
2
and C–H group of phenylene ring form C–HÁ Á ÁO interactions for 30 min at 37 1C show bright green fluorescence in the
À
with the ClO
4
anion with an H-bond distance between 2.04 Å
and 2.32 Å and encapsulate the perchlorate ion perfectly in the
À
cavity (Fig. 4). Upon addition of 3 equiv. of ClO
4
ions to the
–H
solution of PDI 1, the up-field shift of benzimidazolium C
2
and N–CH signals, respectively, by 0.2 and 0.07 ppm in its
2
1
H NMR spectrum (see Fig. S8, ESI†) also supports the binding
À
of the ClO4 anion in the cavity of PDI 1.
For the analytical applications of PDI 1, we have also carried
Fig. 5 Photographs of fluorescence quenching (under 365 nm UV light)
À
out analysis of tap water spiked with various concentrations of
of solution coated TLC strips of PDI 1 for the detection of ClO
4
; (a) TLC
À
ClO
4
. The concentrations thus determined by fluorescence strip with a drop of water; TLC strips on addition of 6 mL of different con-
À À7 À6 À5 À4 À3
4
(b) 10 M (c) 10 M (d) 10 M (e) 10 M (f) 10 M.
centration of ClO
spectroscopy agree with actual concentrations with a relative
2
[
The size of each TLC strip is 1 cm .]
error of 2.7% or better and a RSD below 3.5% (Table S1, ESI†).
À
The PDI 1 was also tested for detection of ClO4 in a sample
solution prepared from flash powder of firework. The response
À
towards ClO4 occurred immediately with the addition of 20 mL
of sample solution to the solution of PDI 1 (10 mM, HEPES
À
buffered–10% DMSO), and the concentration of ClO
4
was
Fig. 6 Images of C6 glioma cells: (a) brightfield image of C6 glioma cells,
b) fluorescence image of C6 glioma cells without PDI 1, (c) fluorescence image
(
À
of C6 glioma cells incubated with ClO
image of C6 glioma cells incubated with PDI 1 (10 mM) for 30 min
4
(40 mM) for 30 min, (d) fluorescence
(
e) fluorescence image of C6 glioma cells incubated with PDI 1 (10 mM)
À
for 30 min and then incubated with ClO
4
(40 mM) for another 30 min and
4À
Fig. 4 The energy minimized structure (DFT 6-31G*) of PDI 1 + ClO
complex.
(f) fluorescence image of C6 glioma cells incubated with PDI 1 (10 mM) for
À
30 min and then incubation with ClO
4
(80 mM) for another 30 min.
13996 | Chem. Commun., 2014, 50, 13994--13997
This journal is ©The Royal Society of Chemistry 2014

View Article Online
Communication
ChemComm
cytoplasmic region upon using 488 nm excitation laser [Fig. 6(d)].
5 (a) L. Schmidt-Mende, A. Fechtenkotter, K. Mullen, E. Moons, R. H.
Friend and J. D. MacKenzie, Science, 2001, 293, 1119; (b) H. Quante,
Y. Greets and K. Mullen, Chem. Mater., 1997, 9, 495.
6 (a) P. Singh, L. S. Mittal, S. Kumar, G. Bhargava and S. Kumar,
J. Fluoresc., 2014, 24, 909; (b) M. Marcia, P. Singh, F. Hauke,
M. Maggini and A. Hirsch, Org. Biomol. Chem., 2014, 12, 7045.
This indicated that PDI 1 is permeable to C6 cells and can be used
À
for bioimaging of ClO4 in live cells. When C6 glioma cells
À
pretreated with PDI 1 were incubated with ClO4 (40 mM) for
another 30 min, bright green fluorescence decreased [Fig. 6(e)].
The bright green fluorescence was further quenched when C6
7
(a) L. W. Yan, L. Yang, J. B. Lan and J. S. You, Sci. China, Ser. B:
Chem., 2009, 52, 518; (b) X. He, H. Liu, Y. Li, S. Wang, Y. Li, N. Wang,
J. Xiao, X. Xu and D. Zhu, Adv. Mater., 2005, 17, 2811.
Glioma cells pretreated with PDI 1 were incubated with a higher
À
8 (a) L. H. Feng and Z. B. Chen, Sens. Actuators, B, 2007, 122, 600; (b) Y.-B.
Ruan, A.-F. Li, J.-S. Zhao, J.-S. Shen and Y.-B. Jiang, Chem. Commun.,
concentration of ClO
4
ions (80 mM) [Fig. 6(f)]. MTT assay using
PDI 1 shows no significant difference in the proliferation of the C6
glioma cells in the absence or presence (98% cell viability) of PDI 1
2
010, 46, 4938; (c) Y. Miyake, H. Togashi, M. Tashiro, H. Yamaguchi,
S. Oda and M. Kudo, et al., J. Am. Chem. Soc., 2006, 128, 2172.
(a) J. Li, Y. Wu, F. Song, G. Wei, Y. Cheng and C. Zhu, J. Mater.
Chem., 2012, 22, 478; (b) B. Wang, F. Wang, H. Jiao, X. Yang and
C. Yu, Analyst, 2010, 135, 1986.
9
(see Fig. S10, ESI†).
In conclusion, PDI 1 could be useful for the detection of
perchlorate ions in aqueous buffer, live rat C6 glioma cells, 10 (a) X. Chen, M. J. Jou and J. Yoon, Org. Lett., 2009, 11, 2181;
(
(
b) X. Guo, D. Zhang and D. Zhu, Adv. Mater., 2004, 16, 125;
c) L. Fan, W. Zhu, J. Li and J. Tian, Synth. Met., 2004, 145, 203.
drinking water and firework flash powder in solution state.
À
In addition, TLC strips doped with PDI 1 could detect ClO
4
1
1
1 X. Feng, Y. An, Z. Yao, C. Li and G. Shi, ACS Appl. Mater. Interfaces,
2012, 4, 614.
2 (a) Z. J. Chen, L. M. Wang, G. Zou, L. Zhang, G. J. Zhang, F. X. Cai
and M. S. Teng, Dyes Pigm., 2012, 94, 410; (b) M. R. Ajayakumar,
P. Mukhopadhyay, S. Yadav and S. Ghosh, Org. Lett., 2010, 12, 2646.
À2
ions 0.8 ng cm
.
This work was supported by the CSIR Grant (No. 02(0084)/
2/EMR-II). We thank DST, New Delhi for FIST programme and
1
UGC, New Delhi, for CAS programme. LSM is thankful to 13 (a) S. P. Kounaves, S. Stroble, R. Anderson, Q. Moore, D. C. Catling,
S. Douglas, C. P. Mckay, D. W. Ming, P. H. Smith, L. K. Tamppari and
A. P. Zent, Environ. Sci. Technol., 2010, 44, 2360; (b) D. S. Richardson,
Anal. Chem., 2009, 81, 4645.
the UGC for SRF fellowship. Live cell experiments were sup-
ported by a grant from DBT, India (BT/IN/German/13/VK/2010)
to VV.
14 T. Zhang, Q. Wu, H. W. Sun, J. Rao and K. Kannan, Environ. Sci.
Technol., 2010, 44, 6947.
1
5 (a) S. M. Otero-Santos, A. D. Delinsky, L. Valentin-Blasini, J. Schiffer
and B. C. Blount, Anal. Chem., 2009, 81, 1931; (b) P. K. Martinelango
and P. K. Dasgupta, Anal. Chem., 2007, 79, 7198.
Notes and references
1
K. Kaur, R. Saini, A. Kumar, V. Luxami, N. Kaur, P. Singh and 16 (a) E. J. Parra, G. A. Crespo, J. Riu, A. Ruiz and F. X. Rius, Analyst,
S. Kumar, Coord. Chem. Rev., 2012, 256, 1992.
2009, 134, 1905; (b) X. Hu, J. Huang, W. Zhang, M. Li, C. Tao and
G. Li, Adv. Mater., 2008, 20, 4074.
2
(a) M. Formica, V. Fusi, L. Giorgi and M. Micheloni, Coord. Chem.
Rev., 2012, 256, 170; (b) J. F. Zhang, Y. Zhou, J. Yoon and J. S. Kim, 17 R. Kumar, S. Kumar, P. Singh, G. Hundal, M. S. Hundal and
Chem. Soc. Rev., 2011, 40, 3416. S. Kumar, Analyst, 2012, 137, 4913.
(a) F. Wurthner, Chem. Commun., 2004, 1564; (b) C. Huang, S. Barlow 18 Z. Chen, V. Stepanenko, V. Dehm, P. Prins, L. D. A. Siebbeles, J. Seibt,
and S. R. Marder, J. Org. Chem., 2011, 76, 2386. P. Marquetand, V. Engel and F. Wurthner, Chem. – Eur. J., 2007, 13, 436.
(a) M. J. Ahrens, M. J. Fuller and M. R. Wasielewski, Chem. Mater., 19 J. Mokac, A. M. Bond, S. Mitchell and G. Scollary, Pure Appl. Chem.,
003, 15, 2684; (b) J. E. Anthony, Chem. Mater., 2011, 23, 583. 1997, 69, 297.
3
4
2
This journal is ©The Royal Society of Chemistry 2014
Chem. Commun., 2014, 50, 13994--13997 | 13997