Highly selective, sensitive and quantitative detection of Hg2+ in aqueous medium under broad pH range

Article abstract of DOI:10.1039/c1cc12018b

Amidothiourea linked acridinedione derivatives selectively detect Hg ²⁺ in unbuffered aqueous solution under broad pH range with both single- and two-photon excitation. The observed linear fluorescence intensity change allows the quantitative d

Full text of DOI:10.1039/c1cc12018b

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COMMUNICATION
Highly selective, sensitive and quantitative detection of Hg2+ in aqueous
medium under broad pH rangew
Ranganathan Koteeswari, Pichandi Ashokkumar, E. J. Padma Malar,*z
Vayalakkavoor T. Ramakrishnan* and Perumal Ramamurthy*
Received 9th April 2011, Accepted 19th May 2011
DOI: 10.1039/c1cc12018b
Amidothiourea linked acridinedione derivatives selectively detect
Hg2+ in unbuffered aqueous solution under broad pH range with
both single- and two-photon excitation. The observed linear
fluorescence intensity change allows the quantitative detection
of Hg2+ in the concentration range of 22 nM–0.33 lM with the
lower detection limit of 2 nM.
Hg2+ probes with good water solubility, high selectivity and
sensitivity are reported,11j,12 detailed investigations on the
optical changes and how individual sensors provide optical
feedback are lacking. As far as quantitative practical Hg2+
detection is concerned, a linear fluorescence response, uniform
fluorescence output at broad pH range, compatibility with
aqueous medium, higher selectivity, sensitivity, fast response
and easy synthetic procedures of probes are most important.
The Hg2+ probes reported so far have achieved only a few of
the above mentioned criteria and do not satisfy all the desired
features.
The development of selective and sensitive detection of toxic
heavy metal ions has attracted considerable attention because
of the wide use of these metal ions and their subsequent impact
on the environment.1 Among these, Hg2+ is considered as
highly dangerous, because both elemental and ionic mercury
can be converted into methyl mercury by bacteria in the environ-
ment, which subsequently bioaccumulates through the food
chain.2 The Environmental Protection Agency (EPA) standard
for the maximum allowable level of inorganic Hg2+ in
drinking water is 2 ppb.3 Several optical methods using organic
fluorophores,4 nanoparticles,5 semiconductors,6 DNAzymes,7
and oligonucleotide8 have been developed for simple and rapid
detection of Hg2+. Among the optical sensors, the fluorescence
based approach is a promising tool for a simple and sensitive
detection of Hg2+ and also for rapid tracking in biological,
toxicological and other environments.9 To date, a number of
functional fluoroionophores exhibiting optical sensing ability
have been developed for the monitoring of Hg2+ ions.10
Another approach, which makes use of a chemodosimeter11
through a specific chemical reaction between dosimeter molecule
and target species to form a coloured or a fluorescent product,
has drawn much attention in recent years. Because of the strong
thiophilic affinity of Hg2+, fluorescence changes associated
with mercury-promoted desulfurization reactions, including
hydrolysis,11a,b cyclizations,11c,d ring opening of rhodamine
spiro systems11e–h and eliminations,11i have been used in the design
of chemodosimeters for Hg2+. Even though a few fluorescent
Recently, two-photon microscopy (TPM) has become a
vital tool in biology.13 Compared to traditional fluorescence
microscopy, TPM offers intrinsic 3D resolution combined
with reduced photo-toxicity, increased specimen penetration,
and negligible background fluorescence. However, two-photon
(TP) probes for Hg2+ are rare.14 In this paper, we report
amidothiourea linked acridinedione (ADD) dosimeters that
exhibit all the important features of Hg2+ detection, including
two-photon excited fluorescence.
Amidothiourea linked ADD dyes (Scheme 1) were synthesized
and characterized by the known procedure.15 These dyes show
the absorption and emission maxima in water in the region
of 390 and 440 nm, respectively, which are assigned to the
intramolecular charge transfer (ICT) from the ring nitrogen
atom to the ring carbonyl centre within the ADD moiety.
Addition of Hg2+ to an aqueous solution of 1a did not lead
to any significant change in the longer wavelength absorption
maximum (Fig. S1aw), which indicates the absence of any
ground-state interaction between the Hg2+ reaction centre and
ADD chromophore. The corresponding fluorescence spectrum
(Fig. S1bw) shows a 92-fold fluorescence quenching, without
any spectral shift. The fluorescence intensity decrease of 1a
was found to limit with the addition of one equivalent of Hg2+
that reveals a 1 : 1 stoichiometry reaction. Unlike other
National Centre for Ultrafast Processes, University of Madras,
Taramani Campus, Chennai-600 113, India.
E-mail: ejpmalar@yahoo.com, vtrk28@yahoo.com,
prm60@hotmail.com; Fax: 091-44-24546709; Tel: 091-44-24547190
w Electronic supplementary information (ESI) available: Experimental
procedures and characterisation of compounds, emission spectra with
the addition of Hg2+. See DOI: 10.1039/c1cc12018b
z Research Scientist C, University Grants Commission, New Delhi,
India.
Scheme 1 Structure of Hg2+ probes.
c
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environmental or biological samples, the fluorescence intensity
should be resistant to changes in pH that may occur in
unbuffered natural systems, or in the analysis of samples from
mildly acidic and/or basic environments. The changes in the
fluorescence intensity were monitored at solution pH values
ranging 3–9, within which most biological samples can be
tested. The emission intensity at 446 nm was greatly reduced in
strongly acidic medium (pH o 3) due to the protonation of the
ADD carbonyl oxygen.17 On the other hand, in highly basic
medium (pH 4 11), fluorescence intensity at 446 nm is
decreased with the formation of a new emission peak at
515 nm, due to the deprotonation of ring amino hydrogen.17
No substantial change in the fluorescence intensity of 1a was
observed in the pH range of 4 to 9 (Fig. 2b). Addition of Hg2+
to this solution caused a similar (ca. 90-fold) fluorescence
quenching. The uniform activity over such a wide range of
pH makes this molecule suitable for the analysis of environ-
mental samples that would occur well within this extended
range of pH, or for use in unbuffered medium.
The high thiophilicity of Hg2+ promoted desulfurization
leading to the formation of dosimetric product, 1,3,4-oxadiazole
(Scheme 2).18 Due to the removal of thiocarbonyl group,
electron density around the aniline moiety is increased, which
promotes an efficient intramolecular through-space photo-
induced electron transfer (PET) from the aniline moiety to
the relatively electron deficient excited state ADD fluorophore.
A similar kind of PET process from methoxybenzene substituted
at the 9th position of ADD is already reported.19 To confirm
the involvement of a PET process, we have synthesized two
more ADD derivatives with strong electron releasing and
withdrawing groups at the PET donating aniline moiety.
Substitution of electron releasing OCH3 group (1b) leads to
a 118-fold fluorescence quenching with the addition of Hg2+
(Fig. S2w) due to the enhanced PET in the dosimetric product 4b,
whereas, compound 1c having electron withdrawing NO2
group shows only 1.6-fold fluorescence quenching (Fig. S3w)
with the addition of Hg2+, due to the diminished PET process
in the product 4c.
Fig. 1 Fluorescence quenching of 1a (0.34 mM) upon addition of
Hg2+ in water–MeOH (99 : 1, v/v), lexc = 385 nm. Inset shows the
changes in the fluorescence intensity at 446 nm upon addition of Hg2+
(0–0.33 mM)
reported dosimetric molecules, probe 1a showed a fluorescence
response towards Hg2+ within 30 s. No further change in
the fluorescence intensity was seen even at prolonged period
(up to 20 min), which allows a rapid real time detection of
Hg2+. For the practical quantitative detection, fluorescence
spectral changes should vary linearly with the concentration of
Hg2+.To test this, we carried out fluorescence titration studies
of 1a (0.34 mM) with Hg2+ (Fig. 1). A linear response of the
fluorescence intensity as a function of [Hg2+] was observed
from 22 nM to 0.33 mM (R2 = 0.999) (inset of Fig. 1). The
observed linearity in the nanomolar concentration range
allows the quantitative detection of Hg2+ in aqueous medium.
The detection limit (DL) can be calculated with the equation,16
DL = 3S0/m, where ‘‘m’’ is the calibration sensitivity of the
fluorescence intensity change (DF = F0 ꢀ F) vs. [Hg2+], and
‘‘S0’’ is the standard deviation of the blank signal (F0) obtained
without Hg2+. From this, the lower detection limit was found
to be 2 nM, which is close to the EPA standard.
Changes in the fluorescence intensity of 1a caused by other
metal ions, including Na+, K+, Mg2+, Ca2+, Sr2+, Ba2+
,
Mn2+, Fe2+, Co2+, Ni2+, Cu2+, Zn2+, Ag+ were also
measured as shown in Fig. 2a. Among the metal ions investi-
gated, Cu2+ and Ag+ show 4.9- and 3.4-fold fluorescence
quenching, respectively, whereas other metal ions did not show
any significant change in the emission intensity even at higher
concentration, when compared to Hg2+. Fluorescence quenching
of 1a with Hg2+ in the presence of each of the above metal
ions (20 mM each) is observed to be similar to that of 1a with
Hg2+ alone (i.e., 92-fold quenching), except for Cu2+ and
Ag+, which show 95.0 and 94.2 fold fluorescence quenching,
respectively. For the rapid monitoring of aqueous Hg2+ in
Involvement of a PET process was further confirmed from
two more ADD derivatives, 2 and 3 which have methyl and
tolyl group, respectively at the PET acceptor moiety. Addition
of Hg2+ to 2 and 3 shows 16- and 20-fold fluorescence
quenching (Fig. S4 and S5w), respectively, which is very low
as compared to that of 1a with Hg2+. The presence of the
electron releasing methyl group at the PET acceptor (ADD
moiety) decreases the extent of PET in the dosimetric product
and results in the decreased fluorescence quenching of 2 with
the addition of Hg2+, as compared to that of 1a with Hg2+
.
Fig. 2 (a) Fluorescence intensity ratio (I0/I) of 1a (3.38 mM) in
water–MeOH (99 : 1) upon addition of different metal ions (5 times
higher concentration than that of Hg2+); (b) Fluorescence intensity
changes of 1a (3.38 mM) (K) and 1a (3.38 mM) + Hg2+ (3.60 mM) (’)
at various pH.
Scheme 2 Hg2+ promoted desulfurization and the formation of
1,3,4-oxadiazole derivative.
c
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Notes and references
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Fig. 3 TPM images of H9C2(2-) cells labelled with 3 (8 mM) upon
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The observed electronic effects and lower fluorescence quantum
yield and shorter lifetime of 4a and 4b (Table S1w) clearly confirm
the operation of PET process in these dosimetric products.
To explore the utility of two-photon excited fluorescence, we
have tested the ability of these molecules to detect Hg2+ by
two-photon excitation. The linear dependence of output fluores-
cence intensity (Iout) on the square of input laser power (mw2)
(Fig. S6w) confirms that this is a two-photon excitation
mechanism. Among all the ADD derivatives, compound 3
shows a relatively higher TPA cross section of 14.6 GM, due to
the increased conjugation from tolyl group. The two-photon
fluorescence excitation spectra (Fig. S7w) of 3 was determined
by the two-photon excited fluorescence (TPEF) method, which
shows a 10 nm blue shift when compared to one-photon spectra,
as observed for many other fluorophores.20 Compound 3 exhibits
a significant TPEF centred at 450 nm. Addition of Hg2+ to 3
leads to a gradual fluorescence intensity decrease (Fig. S8w) as
observed in single photon excitation. Addition of Hg2+ did
not show any significant variation in the TP absorption cross
section; hence the observed quenching in the intensity is only
due to the decreased quantum yield of 3 in the presence of
Hg2+. To demonstrate the potential applications of 3 for TPM
imaging in living cells, H9C2(2-) cells were cultured and
stained with 3 for 15 min, washed with PBS buffer (pH 7.4)
to remove the remaining 3, the treated cells were then incubated
with Hg2+ in culture medium for 15 min. While the cells treated
with only 3 shows bright fluorescence (Fig. 3a), the cells treated
with both 3 and Hg2+ did not show any fluorescence (Fig. 3c).
The fluorescence images clearly indicate that the probe 3 can
detect a minimum of o2 mM of Hg2+ in live H9C2(2-) cells.
We have developed a selective and sensitive Hg2+ probe for
the quantitative detection in aqueous medium. The ability of
this probe to function in unbuffered aqueous solution with
linear and fast fluorescence response will be useful for rapid
and quantitative detection of Hg2+ in environmental samples.
The operation of PET process in the fluorescence signalling
action was confirmed by varying the electron releasing and
withdrawing groups at the PET donor and acceptor moieties.
Even with the lower TPA cross section, we could detect Hg2+
by TPM imaging.
This work was supported by University Grants Commission
(UGC) and Department of Science and Technology (DST),
Government of India. We thank Dr G. Sudhandiran, Dept. of
Biochemistry, University of Madras, for providing cell lines.
R.K. thanks University of Madras for University Research
Fellowship.
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c
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Chem. Commun., 2011, 47, 7695–7697 7697