A CN- specific turn-on phosphorescent probe with probable application for enzymatic assay and as an imaging reagent

Article abstract of DOI:10.1039/c2cc37243f

A new "turn-on" luminescence probe for imaging the uptake of 0.2 ppm inorganic CN^- in live HeLa cells as well as for probing the CN^- generation through an enzymatic process in a virtual aqueous medium at appropriate pH. This journal is

Full text of DOI:10.1039/c2cc37243f

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COMMUNICATION
À
A CN specific turn-on phosphorescent probe with
probable application for enzymatic assay and as an
imaging reagent†
Cite this: Chem. Commun.,
2
013, 49, 255
Received 4th October 2012,
Accepted 30th October 2012
a
a
a
b
b
Upendar Reddy G., Priyadip Das, Sukdeb Saha, Mithu Baidya, Sudip K. Ghosh*
a
and Amitava Das*
DOI: 10.1039/c2cc37243f
www.rsc.org/chemcomm
À
2+
A new ‘‘turn-on’’ luminescence probe for imaging the uptake of of CN towards Cu -ions and the consequential displacement
À
0
.2 ppm inorganic CN in live HeLa cells as well as for probing the methodology is utilized for its recognition in an aqueous
CN generation through an enzymatic process in a virtual aqueous environment. Hydrated CN , which is present predominantly
À
10
À
medium at appropriate pH.
in aq. medium, also attributes to its poor reactivity as a neucleo-
phile toward a chemodosimetric reagent. Thus, the issue of
À
Acute toxicity of the cyanide ion towards mammalians results specific, efficient and instantaneous recognition of CN in aqueous
from its ability to interfere with the electron transport process medium is a challenging one and any new reagent that works
and thus, inhibits the cellular respiration. Despite such a under physiological conditions as well as address these issues has
5
–8,11
detrimental effect on human health, cyanide as a reagent has significance in the area of cyanide detection.
been used widely in various industries. Accumulation of a
1
The fluorescence-based process for probing the binding
higher level of cyanide could also happen through consumption of induced phenomena is generally preferred over others owing
2
certain foods and plants. Its acute toxicity accounts for a very low to the possibility of attaining higher sensitivity as well as
tolerance limit and according to the regulatory bodies (U.S. EPA) developing an imaging reagent for detection of the cellular uptake
the maximum acceptable level of cyanide in drinking water and of the analyte with spatial resolution. In this regard cyclometallated
3
environmental primary standards is 0.2 and 0.5 ppm, respectively.
Ir(III)-complexes have received recent attention due to their better
Prevalent industrial use as well as the increasing threat of its use stability towards photo-bleaching and favourable photophysical
1
2
with malicious intention has actually led to a recent surge of properties. For such Ir(III)-complexes, an efficient spin–orbit
interest for the development of suitable reagents for selective coupling process favours the ISC process and thus helps
À
detection of CN in water or in an aqueous environment. Further, in populating the first excited triplet state having a longer
1
2
the possibility of using such a reagent to develop an enzymatic lifetime. This has been utilized successfully in designing
À
assay for probing the in situ CN generation initiated by an Ir(III)-polypyridyl and Ir(III)-cyclometallated complexes as the
important enzyme like hydroxynitrile lyase (HNL) is an added phosphorescence-based signalling unit as well as an imaging
4
13
motivation and challenge. Several methodologies have been reagent for intracellular studies. Generally, a higher degree of
À
adopted for designing an efficient CN sensor, which include localization of these Ir(III)-complexes in the perinuclear region
recognition through hydrogen bonded adduct formation,
chemodosimetric detection, utilizing the metal–cyanide ion organelles. In the present study, we have used a Ir(III)-based
5
occurs due to interactions of these complexes with hydrophobic
6
13c
7
coordination in a metal ion based receptor, or by adopting the cyclometallated complex having a pendant Cu(II)-moiety (1ÁCu) for
8
À
displacement reaction pathway. However, many such systems recognition of CN in virtual aq. HEPES buffer medium (10 mM
3
suffer from the problem of achieving the specificity either due aq. HEPES buffer–CH CN, 99.6 : 0.4, v/v; pH 7.6) with associated
to the interference of other anions like fluoride, acetate, turn-on phosphorescence response. This reagent could also be
phosphate etc., and/or the desired sensitivity owing to the high used as an imaging reagent for detection of the cellular uptake of
À
À1
9
À
À
solvation energy of CN in water (À339 kJ mol ). The affinity CN ions in HeLa cells from aqueous-buffer media (pH 7.6) with
[
CN ] as low as 0.2 ppm as well as for developing an assay for
a
CSIR-Central Salt & Marine Chemicals Research Institute, Bhavnagar 364002,
Gujarat, India. E-mail: amitava@csmcri.org
probing the in situ release of cyanide from a cyanohydrin by the
HNL enzyme under physiological conditions.
The absorption and photoluminescence spectra of 1 (Scheme 1
and ESI†) were recorded in virtual aq. HEPES buffer (pH 7.6)
medium at room temperature (Fig. 1A). The intense absorption
b
Department of Biotechnology, Indian Institute of Technology, Kharagpur,
West Bengal 721302, India. E-mail: sudip@hijli.iitkgp.ernet.in
Electronic supplementary information (ESI) available: Synthesis,
†
characterization details of L
1
, L
2
, 1 and 1ÁCu, experimental methodologies and
4
À1
À1
sample preparation. See DOI: 10.1039/c2cc37243f
band maximum at 260 nm (e = 5.2 Â 10 mol L cm ) was
This journal is c The Royal Society of Chemistry 2013
Chem. Commun., 2013, 49, 255--257 255

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Scheme 2 Fluorescence responses of 1ÁCu upon treatment with CNÀ.
absence and presence of 40 mole equivalent of various anionic
À
analytes (X ) in buffer media (pH 7.6) were recorded (Fig. 1B) to
examine the luminescence response of this receptor towards
different common and physiologically significant anionic
À
Scheme 1 Molecular structures of L
2
, 1 and 1ÁCu and methodologies that were analytes (Fig. 1B). Among all these anions, only CN showed
adopted for their synthesis.
a turn-on emission response with a maximum at 583 nm (lext
=
380 nm), which was identical to the emission spectra recorded
for complex 1 (Fig. 1B). It is worth mentioning here that no
detectable change in the electronic spectral pattern of 1ÁCu was
À
observed either upon addition of CN or any other anion studied
under identical experimental conditions. Restoration of the lumi-
À
nescence spectra of 1 upon addition of CN to the solution of 1ÁCu
À5
(
2.0 Â 10 M) in buffer medium (pH 7.6) was due to the strong
À
affinity of CN towards the Cu(II)-centre, which eventually led to
decomplexation and formation of Cu(CN)
À(nÀ2)
(n = 2 or 4)
À5
n
Fig. 1 (A) Electronic and luminescence (lext = 380 nm) spectra of 1 (2 Â 10 M).
B) Luminescence spectra (lext = 380 nm) of 1ÁCu in the absence and presence of
(
Scheme 2). This was further confirmed from the ESI-MS studies.†
(
À
À
À
À
À
À
À
À
4À
À
It may be mentioned that the extent of luminescence changes as
various anions (X = F , Cl , Br , I , CN , CH
3
CO
2
, H
2
PO
4
, P
2
O
7
, HSO ,
4
À
À
À
À
À
À
À
NO
3
, NO
2
, N
3
, ClO
4
, PhCO
2
and IO
4
); inset: luminescence titration profile a function of [CN ] was instantaneous and showed a linear
À5
À
À4
À
À5
À4
of 1ÁCu (2.0 Â 10 M) towards varying [CN ] (0 to 8.0 Â 10 M) with
dependency for [CN ] of 0.6 Â 10 to 5.0 Â 10 M. Systematic
Mon
l
ext = 380 nm and l
= 583 nm. All studies were performed in aq. HEPES
titration with 1ÁCu in the buffer solution (pH 7.6) with increasing
buffer–CH
3
CN (99.6 : 0.4, v/v; pH 7.6).
À
[CN ] resulted in the restoration of the original emission spectra
of 1 (inset in Fig. 1B). Thus, 1ÁCu could act as a ‘‘turn-on’’
À
phosphorescence probe in the presence of excess CN .
assigned to typical ppy and L
2
based ligand centered (LC) p–p*
The lowest detection limit of 0.38 ppm was evaluated from
the lower concentration range of the linear dependency of the
emission response at 583 nm (inset, Fig. 1B) in buffer medium of
transitions. Whereas, the less intense absorption shoulder
4
À1
À1
at 311 nm (e = 1.5 Â 10 mol L cm ) was assigned to the
n–p*-based transitions of ppy and L . The relatively weak and
2
À
4
À1
À1
pH 7.6. The [CN ] of 0.38 ppm is lower than the value set by the U.S.
broad band at 380 nm (e = 4.7 Â 10 mol L cm ) was
attributed to a spin allowed dpIr(III) - p*L2/ppy-based metal-to-
ligand charge transfer ( MLCT) transition; while an even weaker
3
EPA for the environmental primary standard. Thus, considering
À
I
the sub-micromolar detection limit for CN under physiological
conditions, the present example is a significant one.
tail at a longer wavelength could be accounted for a dpIr(III)
-
3
13
The switch on luminescence response of 1ÁCu as a function
p*L2/ppy-based MLCT transition. No significant change in the
absorption spectral pattern of 1ÁCu was observed when the
spectrum was recorded under identical conditions.†
À
of [CN ] offered us the opportunity to use 1ÁCu as an imaging
À
reagent for the detection of the cellular uptake of CN in live
Upon excitation at 380 nm, complex 1 exhibited a strong
MLCT emission band with a lmax of 583 nm (Fig. 1A). The
3
relative emission quantum yield (F
medium was found to be 0.038 (using Ru(bpy)
1
) for 1 in buffer (pH 7.6)
2
+
3
as a standard).
The emission spectral pattern of 1 was found to be invariant for
the pH range of 6–8. Photoluminescence spectra recorded for
Mon
1
(
ÁCu (lext = 380 nm and lems = 583 nm) in buffer medium
pH 7.6) revealed that the phosphorescence for 1 was virtually
quenched (F1 = 0.0047) upon formation of 1ÁCu (Fig. 1B) due
ÁCu
2
+
9
14
to the presence of the paramagnetic Cu ion (d system).
Formation of 1ÁCu from 1 was also associated with a detectable
change in solution luminescence from orange red to colourless.
The binding stoichiometry (1 : 1) and formation constant
Fig. 2 (A) Confocal micrographs of live HeLa cells in the presence of 1ÁCu (5 mM in
3
aq. HEPES buffer–CH CN (99.6 : 0.4, v/v; pH 7.6) medium). The phosphorescence
images were acquired after 2 min of treatment of NaCN (aq. HEPES buffer) on
HeLa cells. Bottom panels show an overlay of images with a confocal phase
3
À1
(
8.8 Â 10 M ) were also evaluated for the formation of 1ÁCu.†
À5
Luminescence spectra of the receptor 1ÁCu (2.0 Â 10 M) in the contrast image using FV1000, Olympus.
2
56 Chem. Commun., 2013, 49, 255--257
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In brief, we have reported a unique reagent that is capable of
À
detecting CN either in a pure aqueous environment at a
concentration lower than the safe limit of environmental
primary standards or in live HeLa cells pre-exposed to aqueous
À
solution with [CN ] of 0.2 ppm, the permissible limit set by
the world regulatory body for safe drinking water. Initial
studies clearly reveal that 1ÁCu could be used to develop a
phosphorescence turn-on assay for studying the enzymatic
activity of HNL on a cyanohydrin compound. No such example
was reported earlier using a receptor for cyanide.
A.D. acknowledges DST for funding. S.S. and P.D. thank
CSIR, while U.R.G. and M.B. thank UGC for their fellowships.
S.K.G. thanks DBT for partial support and DST for Confocal
facilities at IIT, Kharagpur, India. A.D. thanks Analytical
Science Department and CIF for help with various analytical
and spectroscopic analysis.
Fig. 3 (A) Emission spectra of (a) 1ÁCu, (b) HNL, (c) 1ÁCu + mdnl + HNL; {(c) À (b)}
was generated after subtracting the spectra of HNL (b) from the spectra of the
solution having 1ÁCu + mdnl + HNL (c) to nullify the emission due to the HNL.
[
{
(
1ÁCu] = 0.02 mM, [mdnl] = 0.8 mM, [HNL] = 10 mL; (B) emission spectra of
1ÁCu (0.02 mM) + mdnl (0.8 mM)} in the absence and presence of varying [HNL]
0–75 mL). Aq. HEPES buffer (pH 6.5) was used for all studies.
HeLa cells (cervical cancer cells) pre-exposed to a buffer
solution (pH 7.6) of 5 mM 1ÁCu. To examine such a possibility, Notes and references
pre-treated live HeLa cells were exposed to various [NaCN]
1
C. Young, L. Tidwell and C. Anderson, Cyanide: Social, Industrial,
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The micrographs of the loaded cells displayed a distinct
increase in the intracellular emission intensity in the red
2
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À
channel (590 Æ 10 nm) with an increase in the [CN ]. Higher
4
À
[CN ] accounted for the regeneration of the luminescent
complex (1) from the nonluminescent binuclear complex 1Á
Cu, which accounted for the enhanced cellular emission. No
significant change of the cell morphology or floating appearance of
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possibility of using this reagent for the intracellular distribution of
CN as low as 0.2 ppm, the [CN ] set by the U.S. EPA for safe
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5
6
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À
only two earlier references that describe the CN detection as low
as 0.013 ppm and 3.25 ppm in 99.95% (0.5%, CH CN) and 99%
3
8b,d
(1% DMSO) aqueous environment, respectively.
The other two
À
articles, which describe the CN detection in pure water medium,
have reported relatively higher detection limits.
We also explored the possibility of using the 1ÁCu reagent for
probing the in situ cyanide generation following an enzymatic
process initiated by HNL, an enzyme that was known to catalyze
the decomposition of mandelonitrile (mdnl) and liberate
11
9
cyanide at pH 6.5, and thus to develop a turn-on assay. Fig. 3 10 (a) Y. Liu, X. Lv, Y. Zhao, J. Liu, Y.-Q. Sun, P. Wang and W. Guo,
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at 437 and 453 nm in aq. HEPES buffer medium of pH 6.5. A
1
distinct increase in emission intensity at B583 nm with an
increase in [HNL] in the solution mixture containing mdnl and
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(
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1
ÁCu suggests an increase in the formation of 1 and thus, the
1
enhanced in situ generation of cyanide species with higher
125, 7377; (b) Y. You and W. Nam, Chem. Soc. Rev., 2012, 41, 7061.
[
9
HNL] (Fig. 3B). We could evaluate the Michaelis constant (K
.99 Â 10 M) from the plot of v (v being the overall rate
=
m
1
3 (a) Y. You, S. Lee, T. Kim, K. Ohkubo, W.-S. Chae, S. Fukuzumi,
G.-J. Jhon, W. Nam and S. J. J. Lippard, J. Am. Chem. Soc., 2011,
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À4
constant) vs. 1/[mdnl] ([mdnl] = 0.5 mM to 50 mM), while [HNL]
(
moderately low value of K
À5
30 mL) and [1ÁCu] (2.0 Â 10 M) were kept unchanged.† A
m
implies that the enzyme has a
moderately high affinity for the substrate mandelonitrile.
14 M. Suresh, A. Ghosh and A. Das, Chem. Commun., 2008, 3906.
This journal is c The Royal Society of Chemistry 2013
Chem. Commun., 2013, 49, 255--257 257