Selective naked-eye detection of Hg2+ through an efficient turn-on photoinduced electron transfer fluorescent probe and its real applications

Article abstract of DOI:10.1021/ac501780z

A simple molecular fluorescent probe 5 has been designed and synthesized by appending anthracene and benzhydryl moieties through a piperazine bridge. The probe upon interaction with different metal ions showed high selectivity and sensitivity (2 ppb) for Hg²⁺ through fluorescence "turn-on" response in HEPES buffer. The significant fluorescence enhancement (～10-fold) is attributable to PET arrest due to complexation with nitrogen atoms of the piperazine unit and Hg²⁺ in 1:2 stoichiometry, in which a naked-eye sensitive fluorescent blue color of solution changed to a blue-green (switched-on). As a proof of concept, promising prospects for application in environmental and biological sciences 5 have been utilized to detect Hg ²⁺ sensitively in real samples, on cellulose paper strips, in protein medium (like BSA), and intracellularly in HeLa cells. Moreover, the optical behavior of 5 upon providing different chemical inputs has been utilized to construct individual logic gates and a reusable combinational logic circuit. The combinational circuit (switch ON mode; OR logic gate) is easily resettable to the original position (switch OFF mode; INHIBIT logic gate) by applying reset chemical inputs (OH^- and PO₄^3-) with great reproducibility.

Full text of DOI:10.1021/ac501780z

Article
pubs.acs.org/ac
Selective Naked-Eye Detection of Hg2+ through an Efficient Turn-On
Photoinduced Electron Transfer Fluorescent Probe and Its Real
Applications
Priyanka Srivastava,† Syed S. Razi,† Rashid Ali,† Ramesh C. Gupta,† Suresh S. Yadav,‡,§
Gopeshwar Narayan,‡,§ and Arvind Misra*,†
†Department of Chemistry, Faculty of Science, Banaras Hindu University, Varanasi 221005 Uttar Pradesh, India
‡Department of Molecular and Human Genetics, Faculty of Science, Banaras Hindu University, Varanasi 221005 Uttar Pradesh, India
S
* Supporting Information
ABSTRACT: A simple molecular fluorescent probe 5 has
been designed and synthesized by appending anthracene and
benzhydryl moieties through a piperazine bridge. The probe
upon interaction with different metal ions showed high
selectivity and sensitivity (2 ppb) for Hg2+ through
fluorescence “turn-on” response in HEPES buffer. The
significant fluorescence enhancement (∼10-fold) is attributable
to PET arrest due to complexation with nitrogen atoms of the
piperazine unit and Hg2+ in 1:2 stoichiometry, in which a
naked-eye sensitive fluorescent blue color of solution changed
to a blue-green (switched-on). As a proof of concept,
promising prospects for application in environmental and
biological sciences 5 have been utilized to detect Hg2+
sensitively in real samples, on cellulose paper strips, in protein
medium (like BSA), and intracellularly in HeLa cells. Moreover, the optical behavior of 5 upon providing different chemical
inputs has been utilized to construct individual logic gates and a reusable combinational logic circuit. The combinational circuit
(switch ON mode; OR logic gate) is easily resettable to the original position (switch OFF mode; INHIBIT logic gate) by
applying reset chemical inputs (OH− and PO43−) with great reproducibility.
real time advantages since they are simple, noninvasive, and
ecently, heavy and transition metal (HTM) contamination
Rhas brought forth serious environmental and health highly sensitive.1 Moreover, progress in development of naked-
problems.1 Among HTMs, mercury (Hg2+) is recognized as a
eye sensitive chemosensors for the detection of HTM ions are
increasingly appreciated important aspect since it may offer
qualitative and quantitative information. Mercury mediated
chemical reactions such as deselenation, hydrolysis, mercura-
tion, and also coordination to sulfur containing receptors are
some common strategies to develop good fluorescent sensors
for mercury;5,6 however, systems capable to detect Hg2+
through enhanced fluorescence are rare.7 While looking for a
good sensor motif, particularly for Hg2+, the major concern is to
achieve good optoelectronic properties, “turn-on” emission, as
well as naked-eye sensitive color changes. In contrast to “turn-
off” motifs, “turn-on” emission is preferred to enhance
detection sensitivity and ease low concentration detection
with negligible background. Moreover, the aqueous medium
compatibility is another limiting factor and the fluorescence
probes to detect Hg2+ in pure or partial aqueous medium are
limited in number.8−13 Thus, the development of facile and
detrimental neurological toxin which is widely distributed in the
environment by various natural processes, industrial releases,
and anthropogenic activities.2,3 The bioaccumulation of such
toxic material in living tissues of human and animal bodies via a
food chain causes mercury poisoning, serious neural disorder,
and diseases like Minamata.1−3 The Environmental Protection
Agency (EPA) has set a 2 ppb maximum tolerable level of
mercury contamination in drinking water.2,3 Therefore,
considerable current interest has arisen to develop selective
and sensitive methodologies for the detection of HTMs owing
to their extensive use and ensuing impact on the environment.
Many reports dealing with the detection of Hg2+ show
fluorescence quenching2 because Hg2+ is known to induce a
spin−orbit coupling effect and is spectroscopically and
magnetically silent (filled d10 orbitals).4 Therefore, the common
analytical techniques such as nuclear magnetic resonance
(NMR) and electron paramagnetic resonance (EPR) are not
adequately competent to detect Hg2+ in environmental and
biological systems. Optical based techniques (fluorescence,
phosphorescence, and chemiluminescence) have some obvious
Received: May 5, 2014
Accepted: August 6, 2014
Published: August 6, 2014
© 2014 American Chemical Society
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efficient methodologies to detect Hg2+ in the environment and
living systems through enhanced emission is a continuing
endeavor among the scientific community, including biologists
and environmentalists.
formazan crystals, obtained by the reduction of yellow
tetrazolium salt (MTT) on metabolically active cells, were
dissolved with the help of DMSO (Sigma). The color was
quantified spectrophotometrically on a microtiter plate reader
(Bio-Rad model 680 microplate reader) at 380 nm excitation.
Each experiment was repeated for three times. The cytotoxic
effect of the probe was assessed through the ratio of the
absorbance of the probe treated sample versus the control
sample. The confocal microscopic images were recorded on an
LSM 510 Meta Confocal microscope (20× Zeiss objective
lens). To collect confocal microscopic images, HeLa cells were
treated separately with a 40 μM solution of probe 5 and 5 +
HgNO3 in the dark and washed twice for 5 min with 1× PBS
buffer (phosphate saline, pH 7.4). Cells were recollected by
centrifugation (at 2000 rpm for 2 min) and mounted on slides
with the help of DABCO.
Keeping in mind the paramount importance of achieving
high affinity and selectivity for Hg2+, we herein report a new
sensor motif to detect the Hg2+ through a fluorescence “turn-
on” response in partial aqueous medium (10 mM; HEPES
buffer pH 7.0; ACN/H2O 3:7, v/v). While synthesizing an
innovative rational synthetic typical sensor motif, we
incorporated anthracene core as a fluorescent signaling unit
along with two benzhydryl moieties and piperazine units as a
suitable ionophore to coordinate Hg2+ selectively. We assumed
that as a soft closed-shell cation Hg2+ has a pronounced affinity
to nitrogen-donor ligands, as in the case of classical azacrown
ethers14 and simple piperazine (pKa, 9.8) unit would promote a
stable 1:2 complexation with mercuric salts in aqueous medium,
without interference of competitive metal ions.14 Second, the
introduction of benzhydryl and anthracene moieties in
conjugation to piperazine will form a more stable hydrophilic
photoinduced electron transfer (PET) chemosensor15 by
acquiring a thermodynamically favorable minimum energy
configuration. As a proof of concept, the molecular probe 5 has
shown high sensitivity to detect Hg2+ in real samples, on
cellulose paper strips, and protein medium like BSA. Intra-
cellular fluorescence imaging of Hg2+ was also realized in HeLa
cells.
RESULTS AND DISCUSSION
■
Probe Design and Photophysical Studies. The
synthetic route adopted for preparation of 5 is shown in
Scheme 1. In a sequence of reactions,16 the benzophenone unit
Scheme 1. (i) NaBH4/Ethanol, (ii) SOCl2/DCM, (iii)
Piperazine/K2CO3/DMF, (iv) Paraformaldehyde/HCl/
Dioxane, and (v) 3/K2CO3/DMF
EXPERIMENTAL SECTION
■
Detection of Hg2+ in Real Contaminated Water
Samples. To accomplish, a real crude water sample (50 mL)
was passed through a microfiltration membrane and the pH of
the sample was adjusted using HEPES buffer. The aliquots of
water samples were mixed with accurately prepared different
concentrations of Hg2+ (0.02, 0.2, 2 μM). The resulting samples
then treated with 5 in HEPES buffer (pH, 7.0) to obtain final
mixtures (3 mL) containing an accurate concentration of probe
5 (10 μM) and Hg2+ ions (0.02, 0.2, 2 μM). The samples were
left for 5 min, and fluorescence spectra were measured in
triplicates.
Live Cell Imaging and Cell Viability Test. HeLa cells
were cultured in Dulbeco’s modified eagle medium (DMEM)
supplemented with 10% fetal bovine serum (FBS), 1%
antibiotics, and antimycotic (Himedia) and incubated overnight
at 37 °C in the environment of CO2 (5%) as per manufacturer’s
protocol. Probe 5 (40 μM) was dissolved in a culture medium
containing 0.1% (v/v) DMSO and incubated into the cells for
30 min at 37 °C. As a control experiment, the cells were
pretreated with PBS or N-methylmaleimide (NMM, 500 μM)
for 1 h, then washed (5 min, two times) with 1×PBS buffer
(pH 7.4) and incubated with 5 (10, 20, 30, 40, 50 μM) in the
cultured media containing 0.1% (v/v) DMSO. After 30 min of
incubation, cell images were acquired under a fluorescence
microscope.
To study the cytotoxicity, HeLa were seeded in a 96-well
plates (5 × 103cells/well) in duplicate and were cultured in
complete DMEM medium. HeLa cells were incubated
separately with different concentrations of probe 5 (10, 20,
30, 40, 50 μM) and probe 5 with HgNO3 (1 μM) for 24 h. To
determine cell viability, the colorimetric metabolic activity assay
was performed by incubating treated cells with 3-(4,5-
dimethylthiazol-2-yl)-3,5-diphenyltetrazolium bromide (MTT)
(5 mg/mL MTT reagent in PBS) for 2 h. The insoluble purple
was first reduced with sodiumborohydride and subsequently
reacted with thionyl chloride to get chloromethyl benzhydryl, 2,
in good yield. Compound 2 was refluxed with piperazine in
DMF to get 3 in 80% yield.16 Anthracene was treated with
paraformaldehyde and hydrochloric acid to get bischlorome-
thylanthracene 4 in good yield (∼38%). Compounds 3 and 4
were reacted under anhydrous conditions in the presence of
K2CO3 in DMF to yield probe 5 (85%) as a light yellow color
powder. The chemical structures were well characterized and
data are given as Supporting Information (see Supportive
Information Figures S1−S7).
The aqueous medium compatibility of probe 5 enabled
photophysical investigation in HEPES buffer (10 mM pH 7.0;
ACN/H2O 3:7, v/v). The electronic transition spectrum of 5
(50 μM) showed typical absorption bands at 395, 374, and 355
nm. Probe 5 (10 μM) at 376 nm excitation displayed very weak
broad emission bands of low quantum yield (Φ = 0.001 with
respect to quinine sulfate) at 402 and 422 nm, which is
attributed to the photoinduced electron transfer (PET) process
(Figure 1). Since the designed probe has nitrogen atoms on the
bridging piperazine unit, it is expected to show variable optical
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suggested high sensitivity and selectivity of probe 5 for Hg2+
ions. Moreover, the metal ions interaction studies were also
performed in PBS buffer (10 mM; pH 7.0) and an acetonitrile−
water gradient system (70%; 3:7; v/v). Probe 5 showed
insignificant change in the emission behavior in PBS buffer,
however, in an ACN−H2O gradient system emission intensity
of 5 was relatively high as compared to emission observed in
HEPES buffer. Also upon interaction with metal ions probe 5 in
ACN−H2O showed almost similar binding behavior with Hg2+
with relatively enhanced emission intensity (∼15%) (Figure
S10 in the Supporting Information). Therefore, it is worth to
mention that probe 5 is suitable to detect Hg2+ in ACN−H2O
and HEPES buffer.
Furthermore, the interference studies have been performed
to ascertain the high affinity of 5 for Hg2+. Upon addition of
tested cations (in excess, 20 equiv) to a solution of probable
complex, 5−Hg2+ and reversibly, addition of Hg2+ to a solution
of 5 containing excess of tested cations both the absorption and
emission spectra showed insignificant change and the color of
solution remained persistent to a blue-green (Figure 1 and
Figure S11 in the Supporting Information). Additionally, we
separately tested the affinity of 5 toward biologically relevant
specific metal ions (Mn+) like Fe2+, Fe3+, Mg2+, Ca2+, Zn2+
under similar condition. Notably, the emission spectra of 5
upon interaction with Mn+ ions revealed insignificant sensitivity
and selectivity. However, a marginal interference (∼5−7%) of
Fe3+ occurred in which emission intensity of 5 + Hg2+
decreased by only 1−1.5% in comparison to Hg2+ (Figure
S12 in the Supporting Information).
Figure 1. Bar diagram shows change in emission intensities of 5 and a
complex, 5−Hg2+ upon interference of tested cations in HEPES buffer
(10 mM; pH 7.0; 70% H2O−ACN). Inset: Emission spectra of 5 and
its complex, 5−Hg2+ upon interference of tested cations.
behavior at different pHs. The pH dependent photophysical
studies were performed in HEPES buffer. Notably, the resulting
absorption and emission spectra of 5 have shown pH
insensitivity in the range 7−14. However, in the acidic medium
(pH 1−6.5), absorption spectra showed marginal hypochro-
micity along with a blue-shift of 3 nm while significant
fluorescence enhancement with a bathochromic shift of 8 nm
(Figure S8a,b in the Supporting Information). This is
attributable to protonation of nitrogen atoms of the piperazine
unit of 5 which restricts the PET (PET-Off). The distribution
of 5 in a broad pH range suggested protonation of 5 below pH
≤ 6.0 while remain unaffected at neutral to alkaline pH (pH ≥
6.0) (inset of Figure S8b in the Supporting Information).
Therefore, 5 can be utilized as an appropriate probe to work at
physiological pH (pH 7.0).
The reversible mode of complexation between 5 and Hg2+
has been realized by the addition of a strong chelating reagent,
EDTA, to a solution of a probable complex, 5−Hg2+. Notably,
the revived emission intensity was found almost close to the
intensity of 5 probably, due to strong binding affinity of EDTA
with Hg2+.17 The observed marginal rise in emission intensity
of 5 upon addition of EDTA (in excess) is attributable to the
acidic nature of EDTA and further addition of Hg2+ in excess
did not reflect any considerable change in the emission
behavior of 5. On repeating this process, the emission behavior
of 5 was found nearly consistent for almost 10 cycles (Figure
S13 in the Supporting Information). The emission titration
experiment was performed to understand the binding affinity of
5 with Hg2+ ions. Notably, upon increasing the concentration of
Hg2+ (0−5 equiv) to a solution of 5, the fluorescence intensity
enhances, “turn-on” gradually, and about ∼10.5-fold maximum
fluorescence intensity observed with a red shift of 2−4 nm
(Figure 2). Job’s plot analysis revealed a 1:2 binding
stoichiometry between 5 and Hg2+ (inset of Figure 2). The
emission titration data was utilized to estimate the association
constant for a 1:2 stoichiometry by the B−H method and was
found to be 1.06 × 1010/M2 (Figure S14 in the Supporting
Information) and accordingly the quantum yield was enhanced
Sensitivity and Selectivity of Probe 5 to Detect Hg2+.
Upon interaction with Hg2+, the absorption spectra of 5
displayed a bathochromic shift of ∼3 nm along with a marginal
decrease in molar absorptivity. In contrast, the emission spectra
of 5 (λex = 376 nm) upon interaction with Hg2+ exhibited
fluorescence “turn-on” response in which relative emission
intensity was enhanced, ∼10.5-fold with a red shift of 2−4 nm
(Figure S9 in the Supporting Information) and the color of
solution “switched-on” to a fluorescent blue-green from a
fluorescent blue color (inset of Figure 2). The other tested
metal ions (such as Na+, K+, Ca2+, Zn2+, Pb2+, Ag+, Cd2+, Co2+,
Ni2+, and Cu2+ (5.0 equiv) failed to exhibit any significant
change in the photophysical behavior of 5. Therefore, this
2+
about ∼13-fold (Φ5+Hg = 0.014). Additionally, the limit of
detection (LOD) of 5 to detect Hg2+ ions was estimated
(Figure S15 in the Supporting Information) in HEPES buffer
by our previously reported method.18−20 The estimated 10 nM
(2 ppb) LOD was found comparable to other reported
methods and well in limit, suggested by EPA. Thus, 5 shows
promise for sensitive and selectivity detection of Hg2+.
Figure 2. Emission titration spectra of probe 5 upon addition of Hg2+
(0−5 equiv) in HEPES buffer (10 mM; pH 7.0; 70% aqueous−ACN).
Inset: change in color of 5 upon interaction with tested cations. Jobs
plot for a 1:2 stoichiometry.
Nature of Interaction. In an endeavor to explain the mode
1
of interaction, the H NMR, FT-IR, and mass spectra of the
probe 5 and its complex, 5 + Hg2+ were analyzed. The multiplet
appeared at δ 8.49 and 7.51 ppm and at δ 7.36−7.16 ppm are
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1
Figure 3. Stacked HNMR spectra of 5 and 5 + Hg2+ in DMSO-d6 and a possible mode of interaction between 5 and Hg2+.
attributable to resonances of anthracene (H1,H4,H5,H8 and
H2,H3,H6,H7) and benzhydryl unit protons of 5, respectively
(Figure 3 and Figure S5b in the Supporting Information).
Similarly, resonances appeared at δ 4.41 and 4.23 ppm have
been assigned to −CH2 (H1′) and −CH (H3″) protons while
the piperazine unit protons (−CH2; H1″ and H2″) appeared at
1
δ 2.54 and 2.27 ppm, respectively. In contrast, the HNMR
spectra of a probable complex, 5 + Hg2+ exhibited significant
Figure 4. (a) Calibration sensitivity plot of 5 for Hg2+ and (b) change
in color of 5 and 5 + Hg2+ solution (a = 0.02, b = 0.2, c = 2 μM) in
HEPES buffer and change in color of 5 and 5−Hg2+ on (c) cellulose
paper strips and (d) silica coated slides.
downfield shifts wherein anthracene unit protons shifted
downfield (Δδ = 0.14 and 0.68 ppm) to appear at δ 8.63 and
8.19 ppm (Figure 3). Similarly, H1′, H3″ and H1″, H2″
resonances shifted downfield (Δδ = 0.97, 0.33 and 0.26, 0.22
ppm) to appear at δ 5.38, 4.56 and 2.80, 2.49 ppm, respectively.
The downfield shifts corresponding to the benzhydryl ring
protons was relatively less (Δδ = 0.01−0.04 ppm). The FT-IR
spectrum of 5 showed characteristic stretching vibration bands
at 2961, 2801, 1598, 1492, 1447 and 1331, 1261, 1136, 1095,
1004, 803, 743, and 704 cm−1 corresponding to C−H, CC,
C−N, and C−H (Ar) functions (Figure S16 in the Supporting
Information). Upon interaction with Hg2+, the C−N stretching
vibration band blue-shifted to appear at 1261, 1144, 1110, and
1088 cm−1. The bands appeared at 636 and 626 cm−1 suggested
about an Hg−N interaction.21 Moreover, the HRMS spectra of
5 (Figure S7b in the Supporting Information) showed a
molecular ion peak, m/z at 707.4067 (calcd 707.4069) which
upon complexation with Hg2+ changed to appear at 1358.2958
(calcd 1358.2961) (Figure S17 in the Supporting Information).
Thus, the observed downfield shifts and typical change in
1HNMR spectral pattern of a complex, 5 + Hg2+ along with
change in FT-IR and HRMS spectra clearly supported the
interaction of probe 5 with Hg2+ through the N atoms of
piperazine units in 1:2 stoichiometry.
quantify recovered Hg2+ contamination in real water samples
within the range of 115 to 92% (Table 1), wherein the color of
Table 1. Estimation of Hg2+ in Real Water Samples
sample concentration
% recovery of Hg2+ from the
sample
(μM)
Hg2+ recovered
0
not detected
0.023 0.014
0.19 0.021
1.84 0.12
0.02
0.2
2
115
95
92
solutions changed from a fluorescent blue to a blue-green,
which was easy to visualize with the naked-eye (Figure 4b).
Thus, it is noteworthy to mention that probe 5 has potential to
detect mercury contamination in the environment with high
sensitivity and selectivity.
Detection of Hg2+ on Paper Strips and Silica Coated
Slides. We also performed experiments for a colorimetric
detection of Hg2+ on solid surfaces, such as paper strips and
silica coated slides, with varying concentrations of probe 5 and
Hg2+ solution (please see the Supporting Information).
Interestingly, the naked-eye sensitive color of paper strips of
probe 5 upon interaction with Hg2+ changed from a light yellow
to light blue green color under UV light (Figure 4c). Similarly,
the probe adsorbed on silica coated slides after interaction with
Hg2+ showed an immediate naked-eye sensitive color change
from a dark blue to a fluorescent blue-green under UV light
(Figure 4d).
Analytical Application of Probe 5. Detection of Hg2+ in
Real Contaminated Water Samples. To validate practical
analytical utility of 5 to determine Hg2+ ion concentration in
real contaminated water samples, we first quantified the
fluorescence of probe 5 (10 μM) in the presence of various
concentrations of Hg2+ ions (0−8 μM), and the corresponding
calibration plot was prepared as the standard curve (Figure 4a).
Considering the possible interference of other components
present in real samples, the level of Hg2+ in real water samples
was determined using the standard addition method.22,23 Prior
to real sample detection when 5 was added directly to the water
samples, no significant fluorescence enhancement occurred.
However, when the emission spectra of treated contaminated
water samples were acquired, the recovery of Hg2+ with respect
to the standard calibration curve was excellent and we could
Affinity of Probe 5 to Detect Hg2+ in Blood Plasma
Protein. The potential applicability of probe 5 to detect Hg2+ in
a biological medium, such as a blood plasma protein (bovine
serum albumin, BSA), has also been studied. Our own research
work and others have shown previously that BSA has high
affinity for Hg2+ ions.18−20,24 The presence of tryptophan
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domains25 in BSA (50 mM NaOAc buffer, pH 6.7) exhibit
strong emission at ∼345 nm (at λex = 278 nm) (Figure S18 in
the Supporting Information). However, upon interaction with
Hg2+ (0−220 μM), about ∼83% fluorescence quenching ocuurs
with an emission maxima at 345 nm (blue-shift, ∼8 nm to
appear at 337 nm) (Figure S16 in the Supporting Information).
In contrast, the emission behavior of 5 remained independent
to even a high concentration of BSA (2 μM) (Figure S19a in
the Supporting Information). Thus, the possibilities such as H-
bonding and/or electrostatic interactions with blood plasma
protein is ruled out. However, addition of Hg2+ (5.0 equiv) to a
solution of 5 + BSA (10 μM probe; 2 μM BSA, pH 7.0)
displayed enhanced turn-on emission (∼10-fold) and was
almost close to the intensity of 5 + Hg2+ without BSA (Figure
S19b in the Supporting Information). Moreover, a preliminary
observation made in the presence of thiol containing amino
acids, such as cysteine (Cys), suggested that the emission
behavior of 5 upon interaction with Cys (100 equiv) before
and/or after the interaction with Hg2+ remained unaffected in
HEPES buffer (Figure S20 in the Supporting Information).
Thus, it is important to mention that probe 5 has the potential
to detect Hg2+ in biological samples.
chosen as an optimum concentration to incubate HeLa cells
separately in the dark. The confocal microscopic images
acquired at 380 nm excitation displayed enhanced green
fluorescence for the incubated HeLa cells with 5 + Hg2+ in
which the relative emission intensity was more intense
compared to HeLa cells with probe 5 (Figure 5, images D, E,
and F).
Furthermore, to address the minimum possibility of
3−
interference of abundantly available PO4 ions studies in live
cells were performed in the presence of some biologically
relevant hard metal ions26 (Mn+) including Hg2+ and as a
control in the presence of a cell permeable transition metal ion
chelator,27 TPEN (N,N,N′,N′-tetrakis(2-pyridylmethyl)-
ethylenediamine). The HeLa cells were incubated separately,
with probe 5 + Mn+ ions (5 μM), 5 + Mn++ Hg2+ (1 μM), and 5
+ Mn++ Hg2+ + TPEN (50 μM) and after proper washing with
1× PBS buffer visualized under a confocal microscope at 380
nm excitation (Figure S21 in the Supporting Information). The
HeLa cells incubated with 5 + Hg2+ + Mn+ displayed relatively
more intense enhanced green fluorescence (Figure S21 in the
Supporting Information, images D and E). However, the
incubated HeLa cells containing metal chelator, TPEN (5 +
Mn++ Hg2+ + TPEN) show diminished emission (Figure S21 in
the Supporting Information, image F). The live cell imaging
experiments clearly showed potential application of probe 5 to
detect Hg2+ with high sensitivity and reversibility (with TPEN)
in biological samples. Moreover, the presence of specific metal
Detection of Hg2+ in Living Cells. To study the ability of
probe 5 to permeate cells and detect Hg2+ in vivo and in
biological samples, live cell imaging experiments have been
performed in HeLa cells through confocal fluorescence
microscopy. The cells were first incubated directly with
different concentrations of 5 (10, 20, 30, 40, 50 μM) and
after proper washing with 1× PBS buffer (phosphate saline, pH
7.4) visualized under a microscope. The observed bright blue
color fluorescence indicated excellent permeability of 5 in HeLa
cells (Figure 5, images A, B, and C).
ions (Mn+) is expected to minimize the interference of PO4
3−
ions inside the live cells.
Response of Probe 5 with Anions. We also checked the
applicability of probe 5 to sense the anions in HEPES buffer.
Probe 5 as such was found insensitive to anion since no
considerable change in fluorescence behavior of 5 occurred
upon interaction with different classes of anions (50 equiv) like
F−, Cl−, Br−, I−, OAc−, CN−, ClO4 , HSO4 , NO3 , and PO4
−
−
−
3−
(Figure S22 in the Supporting Information). However, in
HEPES buffer interaction studies of a complex, 5−Hg2+ showed
3−
high selectivity only for PO4 among the tested anions (5
equiv, sodium salts) (Figure 6), wherein enhanced emission of
Figure 6. (a) Emission spectra and bar diagram of 5−Hg2+ upon
interaction with different anions and (b) emission titration spectra of
5−Hg2+ upon addition of PO43−ions (0−5 equiv) in HEPES buffer.
Figure 5. MTT assay for (a) 5 and (b) 5 + HgNO3. Confocal
fluorescence images show localization of 5 (blue; panel B) and 5−
Hg2+ (green; panel E) in HeLa cells. DIC images of HeLa cells (panels
A and D). Merge fluorescence images for 5 and 5 + Hg2+ (panels C
and F; scale bar, 100 μm).
a complex, 5−Hg2+, diminished almost close to intensity of 5,
probably due to interaction of Hg2+ with PO4 ions.28
3−
Furthermore, it is interesting to mention that intensity of 5
3−
The MTT assay was performed to understand cell viability
and cytotoxic tolerance of HeLa cells corresponding to different
concentration of 5 as well as a complex, 5 + Hg2+. The MTT
assay experiment implicated that more than 50% cells were
viable up to 40 μM concentration of 5 and its complex, 5 +
HgNO3 (Figure 5a,b). Therefore, 40 μM concentration was
remained unaffected upon interaction with PO4 in the
presence of hard metal ions26 (Mn+), while a further addition of
Hg2+ (2.5 equiv) to the same solution led to significant
fluorescence enhancement (Figure S23 in the Supporting
Information). This indicates that the expected binding
3−
reversibility of 5 + Hg2+ by PO4 is inhibited with persistent
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3−
Figure 7. Multiple logic gates and combinational circuit exhibited by 5 upon applying six different chemical inputs of Hg2+, H+, OH−, BSA, PO4
,
and EDTA in HEPES buffer.
enhanced fluorescence and the expected quenching in solution
and live cells would remain dogmatic, consequently.
and In10) truth table (Table S1 in the Supporting Information)
corresponds to a new TRANSFER (output “H”) gate. Now a
combination of two outputs (G and H) outfitted a new OR
gate (output “I”). In this case, the absence of both shows low
fluorescence (output state “0”) while the presence of either or
both generates the high fluorescence (output state “1”) (Figure
7). Moreover, we could reset output emission of our resultant
combinational circuit to the low fluorescence state by applying
two reset keys (as OH− and PO43−). When the reset key in the
form of OH− is applied as input along with output “I” of OR
gate led to a TRANSFER gate (output “J”), as the OH−
neutralize the effect of H+ but since Hg2+ is present, the
emission of probe 5 remained in the switched ON-state.
Logic Interpretation. Recently, researchers are trying to
mimic the function of silicon chips and building electronic
devices based on optoelectronic responses of organic/inorganic
molecules in the presence of external stimuli.29,30 The
molecular scaffolds that displays sensitive optical behavior
(output) and transform macroscopic properties according to
logic operations in the presence of chemical/photonic inputs
are in high demand to develop tunable miniaturized electronic
devices, fluorescent logic gates and arranged logic circuits.31−36
The functional fluorescent molecular scaffolds that enable
multiple 2-bit Boolean logics from the same initial state are
limited in number,37 and the combination of individual multiple
logic systems for their possible use as a unimolecular system has
great futuristic applications, particularly in the field of molecular
switches.38,39 Moreover, the reset capacity of functional organic
molecules with arithmetic functions stimulate further potential
to create a low-dimensional computing devices.40
3−
Interestingly, utilizing output “J” as input along with PO4
reset the circuit to original low emission switched-OFF state
(turn-ON → turn-OFF) and regenerate an INHIBIT gate
(output “K”). Now the constructed combinational circuit based
on the emission behavior of 5 is ready for further use (Figure
7). It is important to mention that we could perform the set-
reset operation 3−5 times without considerable loss in emission
properties of the probe 5 under the condition.
To create logic circuits according to the truth tables, digital
values has been assigned as binary numerals, “1” and “0” to
illustrate high and low emission outputs as well as applied
inputs (present 1; absent 0), respectively. Comparatively, 75%
of the maximum output emission intensity was chosen as a
threshold value. Notably, 5 upon applying chemical inputs of
Hg2+ and/or H+ ion exhibited a maximum emission intensity
(high output). In a truth table, outputs emission which are 75%
or above the maximum has been considered high (1) and low
(0), respectively. The optoelectronic properties of 5 displayed
ON-OFF switching behavior in the presence of applied
CONCLUSION
■
Conclusively through this contribution we have demonstrated
the design and synthesis of an efficient photoinduced electron
transfer (PET) fluorescent probe 5. Probe 5 has shown good
real time fluorescence “turn-ON” response, high sensitivity, and
considerable selectivity to detect Hg2+ in partial aqueous
medium, on paper strips, silica coated microslides, and in real
water samples. Probe 5 has shown high cell permeability and
low toxicity to detect Hg2+ sensitively in vivo as well as in
protein medium. The high limit of detection (2 ppb) with
recovery in the range of 90−115% has predicted the potential
application of 5 to sense Hg2+ in biological and environmental
samples. Moreover, the excellent optoelectronic properties of 5
providing different chemical inputs has been utilized to create a
reusable combinational logic circuit and suitable optoelectronic
devices such as molecular logic switches.
chemical inputs, such as PO4 (In1), Hg2+ (In2), H+ (In3),
3−
EDTA (In4), OH− (In5), and BSA (In6), and the respective
truth tables (inset of Figures S24−S29 in the Supporting
Information) corresponds to different logic gates as INHIBIT,
TRANSFER, and OR under respective conditions.
Further, an arranged combinational logic circuit has been
constructed by the combination of outputs of different logic
gates in a unimolecular system (Figure 7). A combination of
outputs (A and B) of two logic gates INHIBIT and OR as
inputs (In7 and In8) gave a new TRANSFER gate (output “G”)
as the absence of both inputs or presence of only In7 exhibit
low emission while the presence of both or only In8 give high
output emission. Similarly, when the outputs (C and D) of two
gates TRANSFER and INHIBIT were utilized as inputs (In9
8698
dx.doi.org/10.1021/ac501780z | Anal. Chem. 2014, 86, 8693−8699

Analytical Chemistry
Article
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ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental details, synthesis, 1HNMR, 13CNMR, FT-IR, ESI-
MS, UV−Vis, and fluorescence spectra. This material is
available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Phone: +91-0542-2307321 ext. 104; +91-0542-6702503. Fax:
+91-0542-2368127; +91-0542-2368175. E-mail:
arvindmisra2003@yahoo.com; amisra@bhu.ac.in.
■
Notes
(33) Pathak, R. K.; Dessingou, J.; Hinge, V. K.; Thawari, A. G.; Basu,
S. K.; Pulla Rao, C. Anal. Chem. 2013, 85, 3707−3714.
(34) Pu, S.; Ding, H.; Liu, G.; Zheng, C.; Xu, H. J. Phys. Chem. C
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The authors declare no competing financial interest.
§The authors have performed experiments in live cells.
ACKNOWLEDGMENTS
■
(35) Raymo, F. M. Adv. Mater. 2002, 14, 401−414.
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The authors are thankful to CSIR, New Delhi, India for funding
and fellowships (SRF to P.S., S.S.R., R.A. and UGC-JRF to
R.C.G.) to carry out the research work. We are also thankful to
the Head of the Department of Chemistry, Faculty of Science,
Banaras Hindu University for creating the necessary facilities to
carry out the advanced research studies.
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