A highly sensitive fluorescent probe for detection of hydrazine in gas and solution phases based on the Gabriel mechanism and its bioimaging

Article abstract of DOI:10.1039/c6ra14212e

A new probe 2-benzo[1,2,5]thiadiazol-4-yl-isoindole-1,3-dione (BTI) based on the Gabriel reaction mechanism was synthesized and characterized for the specific detection of hydrazine with high selectivity against other amines in an organo-aqueous solution. Upon hydrazinolysis of BTI in the presence of hydrazine in a H₂O-DMSO (4:6, v/v) solution (10 mM HEPES buffer, pH 7.4) at room temperature, the chemosensor produces fluorescent aminobenzthiadiazole with a maximum emission at 498 nm along with a color change from colorless to green, allowing selective colorimetric and fluorometric detection of hydrazine by the naked eye. Probe BTI was also successfully applied in vapor phase hydrazine detection into a solid state over other interfering volatile analytes. Furthermore, the probe BTI coated with silica gel TLC plates could act as a visual and fluorimetric probe for hydrazine vapor detection. The experimental detection limit of hydrazine is 2.9 ppb, which is well below the accepted limit (10 ppb) for hydrazine set by the U.S. Environmental Protection Agency (EPA). DFT and TDDFT calculations were performed in order to demonstrate the sensing mechanism and the electronic properties of probe and hydrazinolysis products. Additionally, probe BTI could also be applied for the imaging of hydrazine in living cells.

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ARTICLE TYPE
A highly sensitive fluorescent probe for detection of hydrazine in gas
and solution phase based on the Gabriel mechanism and its bioimaging
Rajkishor Maji,a Ajit Kumar Mahapatra*,a Kalipada Maiti,a Sanchita Mondal,a Syed Samim Ali,a
Prithidipa Sahoo,b Sukhendu Mandal,c Md Raihan Uddin,c Shyamaprosad Goswami,a Ching Kheng
Quahd and Hoong-Kun Fund,e
5
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
DOI: 10.1039/b000000x
A new probe 2ꢀbenzo[1,2,5]thiadiazolꢀ4ꢀylꢀisoindoleꢀ1,3ꢀdione (BTI) based on the Gabriel reaction mechanism was synthesized and
characterized for the specific detection of hydrazine with high selectivity against other amines in oganoꢀaqueous solution. Upon
10 hydrazinolysis of BTI in the presence of hydrazine in H2O–DMSO (4:6, v/v) solution (10 mM HEPES buffer, pH 7.4) at room
temperature, the chemosensor produces fluorescent aminobenzthiadiazole with a maximum emission at 498 nm along with a color
change from colorless to green, allowing selective colorimetric and fluorometric detection of hydrazine by the naked eye. Probe BTI was
also successfully applied in vapor phase hydrazine detection into a solid state over other interfering volatile analytes. Furthermore, the
probe BTI coated with silica gel TLC plates could act as a visual and fluorimetric probe for hydrazine vapor detection. The experimental
15 detection limit of hydrazine is 2.9 ppb, which is well below the accepted limit (10 ppb) for hydrazine set by the U.S. Environmental
Protection Agency (EPA). DFT and TDDFT calculations were performed in order to demonstrate the sensing mechanism and the
electronic properties of probe and hydrazinolysis product. Additionally, probe BTI could also be applied for the imaging of hydrazine in
living cells.
20
45 coulometry,13
potentiometry,14
titrimetry,15
capillary
Introduction
electrophoresis16 and electroanalysis.17 Spectrophotometry using
colored derivatives, such as pꢀdimethylaminobenzaldehyde18 and
chlorosalicylaldehyde,19 are also used to detect hydrazine.
However, these methods are not only complex and time
50 consuming, but also impractical for in vivo hydrazine analysis
because of their postꢀmortem processing and destruction of
tissues and cell contents. Among several detection strategies,
fluorescent techniques are extremely attractive due to their high
sensitivity, low cost, easy implementation, and realꢀtime
55 detection.20ꢀ21 Till now, only a few fluorescent chemodosimeters
for hydrazine have been reported, and almost all of them were
designed based on the deprotection or chemical transformation of
a protecting group by a specific deprotecting agent or analyte.22ꢀ26
For example, the fluorescent sensing system developed by Chang
60 et al.,27 showed the selective deprotection of levulinated
coumarin in presence of hydrazine in DMSOꢀwater. The sensing
system reported by Peng et al.,28 showed a ratiometric hydrazineꢀ
selective NIR probe based on cyanine dye via deprotection of
acetyl group in aqueousꢀorganic solvent. Therefore, it still
65 remains a challenge to develop effective hybrid fluorescent
probes with suitable reactive zone that can act as good
chemodosimeter for the recognition of molecular species, though
such systems are limited in literature in case of hydrazine
Much attention has been focussed in the last few years on the
development of small molecular fluorescent probes for hydrazine
(H2NꢀNH2) because of its wide application and toxicity.1
25 Hydrazine and its derivatives are commonly known fuels in
explosives, antioxidants, rocket propellants, blowing agents,
photographic chemical, corrosion inhibitor, insecticides, and
plant growth regulators.2ꢀ4 In spites of these, it is commonly
known as a neurotoxin, carcinogenic, mutagenic, hepatotoxic and
30 very harmful to human life.5ꢀ6 It is highly toxic and easily
absorbed by oral, breathing routes of exposure or even introduced
to the skin, which mostly affect the lungs, kidney, liver and
central nervous system of living organisms. Moreover, it can
seriously affect the reproductive system in animals after the
35 hydrazine inhalation. It has also been identified as a carcinogenic
agent by U.S. Environmental Protection Agency (EPA) and the
estimated inhalation unit risk is 4.9 × 10−3( µg/m3)−1. Hence,
hydrazine has been classified as a carcinogenic substance by the
same agency with allowable threshold limit value (TLV) of 10
40 ppb,7ꢀ8 which provides an incentive to research for new analytical
approaches capable of determining trace level of hydrazine.
Various traditional analytical techniques for the detection of
hydrazine are available, including electrochemical analysis9 and
chromatography,10 including gas chromatography,11 HPLC,12
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sensing. Furthermore, fewer sensors have been applied to vapor
Experimental Section
29ꢀ30
sensing.
chemodosimeter which employs
Cui et al29 have reported an efficient fluorescent
naphthalic anhydride
a
General Information and Materials
fluorophore. It senses hydrazine in an elegant manner by the
Gabriel reaction. Keeping this in mind, we have envisioned a
chemodosimeter which contains benzothiadiazole fluorophore
which incorporates excellent turnꢀon fluorescence properties
along with enhanced bioꢀcompatibility. Some of the current
probes could only be utilized at low pH (pH ＜ 5) conditions31ꢀ32
Unless otherwise mentioned, materials were obtained from
60 commercial suppliers and were used without further purification.
1H and 13C NMR spectra were recorded on a Brucker 400 MHz
instrument. For NMR spectra, CDCl3 and DMSOꢀd6 were used as
solvent using TMS as an internal standard. Chemical shifts are
expressed in δ ppm units. UV–visible and fluorescence spectra
65 measurements were performed on a JASCO V530 and a Perkin
Elmer LS55 spectrofluorimeter respectively. Single crystal Xꢀ
raydiffraction data for BTI were collected on Bruker
SMART APEX II DUO CCD areaꢀdetector diffractometer at 100
K temperature. Elemental analysis of the compounds was carried
70 out on PerkinꢀElmer 2400 series CHNS/O Analyzer. Chemicals
and solvents used for the synthesis of receptor were purchased
from Sigma Aldrich Chemical Co. (USA) and used without
further purification. Salts of different cations, anions and amines
were purchased from Spectrochem Pvt Ltd. (India). The
75 following abbreviations are used to describe spin multiplicities in
1H NMR spectra: s = singlet; d = doublet; dd = double doublet; t
= triplet.
5
10 which would limit their application in physiological conditions.
Thus, developing a new fluorescence method of monitoring
hydrazine in living cells or sensing vapor phase hydrazine
remains a significant challenge.
In continuation of our research work in the development of
15 various fluorescent chemosensors for important toxic analytes,
herein, we disclose the design and synthesis of a fluorescence
sensor based on phthalimide–benzothiadiazole molecular hybrid,
which can selectively detect hydrazine in aqueousꢀDMSO media.
The selection of the hybrid phthalimide–benzothiadiazole
20 platform is due to favorable photophysical properties of
aminobenzothiadiazole including high quantum yields, high
extinction coefficient, and emission maximum beyond 450 nm in
the visible region. We recently reported the selective detection of
biothiols and hydrazine using benzothiadiazole, BODIPY–pyrene
25 and carbazole based chemosensor.33ꢀ35 Therefore, it is of prime
interest to develop phthalimide–benzothiadiazoleꢀbased hybrid
reactive molecular systems to provide better sensitivity and
selectivity toward sensing of toxic molecules. However, a
benzothiadiazoleꢀbased fluorescent probe for hydrazine detection
30 has not been widely reported. To our knowledge, there are very
few reports36ꢀ38 of selective hydrazine fluorescent probes based
on Gabriel phthalimide type moiety. It is important to note that
our new probe, 2ꢀbenzo[1,2,5]thiadiazolꢀ4ꢀylꢀisoindoleꢀ1,3ꢀdione
(BTI) is highly sensitive to hydrazine with detection limit of
35 8.47×10–8 M.
General method of UV-vis and fluorescence titration
For UVꢀvis and fluorescence titrations, stock solution of sensor
80 BTI was prepared (c = 1 x 10ꢀ6 MLꢀ1) in H2O–DMSO (4:6, v/v)
solution (10 mM HEPES buffer, pH 7.4).The solutions of the
guest cations, anions and primary amines in the order of 1 x 10ꢀ5
MLꢀ1 were also prepared in H2O–DMSO (4:6, v/v) solution (10
mM HEPES buffer, pH 7.4). The test solution of sensor BTI was
85 prepared by proper dilution method. The spectra of these
solutions were recorded by means of UVꢀvis and the fluorescence
methods. All the solvents were purchased from local suppliers
and were distilled by standard procedure before use.
Fluorescent hydrazine probe has been constructed by exploiting
the high nucleophilic reactivity of the hydrazine molecule. In this
work, we judiciously designed probe BTI (Scheme 1) as a new
type of selective hydrazine fluorescent probe based on the
40 Gabriel type hydrazinolysis of benzothiadiazol derivative of
phthalimide. Probe BTI contains a aminobenzothiadiazole moiety
which acts as fluorescent signal transducer and a phthalimide
moiety for reacting zone. It is known that in Gabriel method Nꢀ
substituted phthalimide reacts with hydrazine via simultaneous
45 substitutionꢀelimination process twice gives phthalhydrazide and
free primary amine. Due to a photoinduced electron transfer
(PET) process from the electron donor, fluorophore to the
electron accepter phthalimide (isoindoleꢀ1,3ꢀdione) moiety, probe
BTI is nonꢀfluorescent (Φ = 0.067). However, when treated with
Determination of quantum yield
90 For measurement of the quantum yields of BTI and BTIꢀN2H4,
we recorded the absorbance of the compounds in DMSO solution.
The emission spectra were recorded using the maximal excitation
wavelengths, and the integrated areas of the fluorescenceꢀ
corrected spectra were measured. The quantum yield of BTI and
95 BTIꢀN2H4 were then calculated by comparison with anthracene
(Φs = 0.28 in EtOH) as reference using the following equation.
2
ϕx = ϕs ( Fx / Fs)( As / Ax)(nx / ns2)
Where, X & S indicate the unknown and standard solution
respectively, ϕ = quantum yield, F = area under the emission
100 curve, A = absorbance at the excitation wave length, n = index of
refraction of the solvent.
50 hydrazine, it exhibits
a relatively rapid, timeꢀdependent
enhancement of its fluorescence signal (Φ = 0.704). Such finding
suggests that hydrazine selectively reacts with phthalimide
moiety in probe BTI, thus eliminating PETꢀinduced fluorescence
quenching and we should observe a substantial fluorescence turnꢀ
55 on response due to free aminobenzothiadiazole moiety.
Cell Culture
Vero cell (very thin endothelial cell) (Vero 76, ATCC No CRLꢀ
1587) lines were prepared from continuous culture in Dulbecco’s
2
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modified Eagle’s medium (DMEM, Sigma Chemical Co., St.
following formula: (mean OD in treated wells / mean OD in
Louis, MO) supplemented with 10% fetal bovine serum 55 control wells) X 100.
(Invitrogen), penicillin (100 ꢁg/mL), and streptomycin (100
Synthesis of Compound (BTI)
ꢁg/mL). The Vero 76 were obtained from the American Type
Culture Collection (Rockville, MD) and maintained in DMEM
containing 10% (v/v) fetal bovine serum and antibiotics in a CO2
incubator. Cells were initially propagated in 75 cm2 polystyrene,
filterꢀcapped tissue culture flask in an atmosphere of 5% CO2 and
95% air at 37°C in CO2 incubator. When the cells reached the
10 logarithmic phase, the cell density was adjusted to 1.0 x 105
per/well in culture media. The cells were then used to inoculate in
a glass bottom dish, with 1.0 mL (1.0 x 104 cells) of cell
suspension in each dish. After cell adhesion, culture medium was
removed. The cell layer was rinsed twice with phosphate buffered
15 saline (PBS), and then treated according to the experimental
need.
5
A mixture of 4ꢀaminoꢀ2,1,3ꢀbenzothiadiazole (2) (377.98 mg, 2.5
mmol) and phthalic anhydride (1) (300 mg, 2.03 mmol) in glacial
acetic acid (10 mL) was heated to reflex with stirring for 12 hours
60 in a nitrogen atmosphere. The suspension was poured into iceꢀ
water (50 mL) and filtered to give solid residues. Then the
residues were purified by column chromatography (silica gel,
10% EtOAc in hexane) to afford compound BTI as a pale yellow
solid (yield 611.8 mg, 87% with respect to 4ꢀaminoꢀ2,1,3ꢀ
65 benzothiadiazole). Mp above 250 °C. 1H NMR (CDCl3, 400
MHz) δ(ppm): 8.1178 (d, 1H, ArꢀH, J= 8.8 Hz), 8.0116 (dd, 2H,
ArꢀH, J= 5.32 Hz, 3.04 Hz), 7.8335 (dd, 2H, ArꢀH, J= 5.32 Hz,
3.04 Hz), 7.7391 (t, 1H, ArꢀH, J= 7.2 Hz), 7.6262 (d, 1H, ArꢀH,
J= 7.08 Hz). 13C NMR (DMSOꢀd6, 100 MHz) δ(ppm): 167.42,
70 155.85, 151.98, 136.09, 132.43, 131.08, 130.65, 124.76, 123.27,
117.08. ESI LCMS, m/z = 282.1 [M+1]+; calculated, 281.2894
Anal. Cald for C14H7N3O2S ; C, 59.78; H, 2.51; N, 14.94; S,
11.39 Found: C, 59.94; H, 2.35; N, 14.97; S, 11.36%.
Cellular Imaging Methodology
For fluorescence imaging studies Vero cells, 1 x 104 cells in 1000
ꢁL of medium, were seeded on sterile 35 mm Petri dish, glass
20 bottom culture dish (ibidi GmbH, Germany), and incubated at 37
°C in a CO2 incubator for 10 hours. Then cells were washed with
500 ꢁL DMEM followed by incubation with final concentration
of 1.0 x 10ꢀ6 M BTI probe dissolved in 500 ꢁL DMEM at 37°C
for 1 h in a CO2 incubator and observed under an Olympus IX71
25 microscope. Images analyzed by Imageꢀpro plus (version 6.3)
with excitation at nearby 405 nm FITC filter, and emit at nearby
513 nm. The cells were again washed thrice with phosphate
buffered saline PBS (pH 7.4) to remove any free BTI probe and
incubated in PBS containing NH2ꢀNH2 to a final concentrations
30 of 1.0 x 10ꢀ5 M, incubated for 30 min followed by washing with
PBS three times to remove excess NH2ꢀNH2 outside the cells and
images were captured.
75 Results and discussion
The chemosensor molecule, BTI, was readily synthesized in one
step based on the methodology39 outlined in Scheme 1, and the
structure of the probe was characterized by H NMR, 13C NMR,
1
LCꢀMS (Figure S1ꢀS3 in the Supporting Information) and singleꢀ
80 crystal XRD analysis (Figure 1).
O
NH2
O
glacial acetic
acid
N
N
N
N
Cytotoxicity Assay
O
S
N
85
S
reflex, 12 h
O
O
The cytotoxic effects of probe BTI and BTIꢀN2H4 were
35 determined by an MTT [3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀ
diphenyltetrazolium bromide] assay following the manufacturer’s
instruction (MTT 2003, SigmaꢀAldrich, MO). Vero cells were
cultured into 96ꢀwell plates (104 cells per well) for 24 h. After
overnight incubation, the medium was removed, and various
40 concentrations of BTI, N2H4 and BTIꢀN2H4 (0, 5, 20, 30 and 50
ꢁM) made in DMEM were added to the cells and incubated for
24 h. Control experiments were set with DMSO, cells without
any treatment and cellꢀfree medium were also included in the
study. Following incubation, the growth medium was removed,
45 and fresh DMEM containing MTT solution was added. The plate
was incubated for 3−4 h at 37°C. Subsequently, the supernatant
was removed, the insoluble colored formazan product was
solubilized in DMSO, and its absorbance was measured in a
microplate reader (PerkinꢀElmer) at 570 nm. The assay was
50 performed in triplicate for each concentration of BTI, N2H4 and
BTIꢀN2H4. The OD value of wells containing only DMEM
medium was subtracted from all readings to get rid of the
background influence. The cell viability was calculated by the
1
2
BTI
Scheme 1. Schematic Representation of Synthesis of Compound
90 BTI.
Indeed, as designed, probe BTI is essentially weakꢀfluorescent (Φ
= 0.067) in the neutral aqueous conditions (H2O–DMSO (4:6,
v/v) solution (10 mM HEPES buffer, pH 7.4), whereas
aminobenzothiadiazol 2 is highly fluorescent (Φ = 0.704) around
95 498 nm in the same aqueous buffer. Thus, it is apparent that
compound BTI is promising as a fluorescence turnꢀon probe for
hydrazine provided that compound BTI could be converted by
hydrazine to give fluorescent free amine 2.
A summary of the crystallographic data is given in Table S1 in
100 the Supporting Information. The BTI molecule, Figure 1a, is
twisted with a dihedral angle between the benzothiadiazole
(S1/N2/N3/C10ꢀC14, r.m.s deviation = 0.009 Å) and isoindole
ring systems (N1/C1ꢀC18, r.m.s deviation = 0.022 Å) is
51.61(5)°. In the crystal (Figure 1b), the molecules are linked by
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intermolecular C—H···O and C—H···N hydrogen bonds (Table 55 to πꢀπ* as well as vibronic transitions. Upon the addition of
S2 in the Supporting Information) and resulting in twoꢀ
dimensional planes parallel to (011) (Figure 1b). The molecular
structure is further stabilized by weak aromatic π–π stacking
interactions between the benzene and thiadiazole rings of
hydrazine, the dual absorption bands gradual decreases with a
concomitant growth of a new structureless band at 395 nm,
therefore induce a color change from colorless to yellow (Figure
2). The presence of clear isosbestic point at 362 nm implies that it
5
adjacent molecules [centroid–centroid separation = 3.7002(10) 60 transforms quantitatively to a new species.
Å].
The probe BTI exhibit very weak emission at ∼498 nm when
excited at 313 nm in H2O–DMSO (4:6, v/v) solution (10 mM
HEPES buffer, pH 7.4).
a)
b)
a)
65
b)
BTI
+
N2H4
BTI
10
Figure 1. (a) ORTEP diagram for the stereo view of the
molecular structure as obtained from the single crystal XRD of
15 BTI. (b) The crystal packing, viewed along the b axis, showing
the molecules are linked into twoꢀdimensional planes.
70 Figure 3. (a) Fluorescence spectra (excitation at 313 nm) of
sensor BTI (c = 1 x 10ꢀ6 MLꢀ1) in H2O–DMSO (4:6, v/v) solution
(10 mM HEPES buffer, pH 7.4) in the presence of 0 – 1.5 equiv.
of hydrazine. Inset: the fluorescence change of the sensor BTI
with the addition of hydrazine. (b) Relative fluorescence
75 responses of sensor BTI (c = 1 x 10ꢀ6 MLꢀ1) in H2O–DMSO (4:6,
v/v) solution (10 mM HEPES buffer, pH 7.4) to hydrazine (1.5
equivalents) and other various representative species and primary
amines (50 equivalents each): (1) blank; (2) hydrazine; (3)
Refinement parameters of BTI (Cambridge Crystallographic
Data Centre as entry CCDC 1479814):
20
The sensitivity of BTI toward different amines and their
preferential selectivity toward hydrazine over the other amines
has been studied by absorption and fluorescence titrations. We
found that the addition of trace amounts of hydrazine causes the
25 absorption and fluorescence signal to change rapidly, which is
very important for realꢀtime detection. Therefore, the following
titration experiments were carried out after adding varying
concentrations of hydrazine (Figure 2) to a fixed concentration of
BTI in H2O–DMSO (4:6, v/v) solution (10 mM HEPES buffer,
30 pH 7.4).
ethylenediamine;
(4)
1,3ꢀdiaminopropane;
(5)
oꢀ
80 phenylenediamine; (6) ammonia; (7) hydroxylamine; (8)
cysteine; (9) homocysteine; (10) urea; (11) thiourea;(12) mixture
[BTI+analytes (tested amines) +N2H4]. Inset: the relative
fluorescence intensity (I/I0) as a function of [N2H4]/[ BTI] mole
ratio.
b)
a)
85 Addition of increasing concentrations of hydrazine to the probe
BTI results in the enhancement of fluorescence intensity at 498
nm as a function of the added hydrazine concentration, and the
fluorescence enhancement at 498 nm was up to 9.05ꢀfold (Figure
3). Furthermore, the introduction of hydrazine turned the visual
90 emission of the probe BTI solution from dark to bright green
(Figure 3a, inset), which further supports the fluorescence turnꢀon
response.
BTI
+
BTI
N2H4
35
40
Figure 2. Changes in the UV/vis absorption spectra of sensor
BTI (c = 1 x 10ꢀ6 MLꢀ1) in H2O–DMSO (4:6, v/v) solution (10
mM HEPES buffer, pH 7.4) (a) in the presence of hydrazine (1.5
equivalents) Inset: the relative absorbance (A/A0) as a function of
The changes in the fluorescence spectrum stopped when the
amount of added hydrazine reached 1.5 equivalent of the probe.
95 A plot of fluorescence intensity as
a function of added
[Hydrazine]/[BTI] mole ratio (Figure 3b, inset) shows
a
45 [N2H4]/[ BTI] mole ratio and (b) other representative cations
stoichiometry of 1:1 between the probe and hydrazine and the
intensity goes to highest value at ≥1 equiv. A linear relationship
was observed between the fluorescence intensity and hydrazine
100 amount in the range of 0.025–1.5 ꢁM. The detection limit of
(Cu2+, Hg2+, Zn2+, Cd2+, Mg2+, Fe3+, Al3+ and Ag+), anions (F−,
−
Cl−, Br−, I−, CN−, PO43−, NO3 , S2− and SO42−) and primary
amines
phenylenediamine,
(ethylenediamine,
ammonia,
1,3ꢀdiaminopropane,
hydroxylamine, cysteine,
oꢀ
probe BTI towards hydrazine was calculated to be 8.47×10–8
M
50 homocysteine, urea and thiourea) (50 equivalents each) Inset:
color changes of the sensor BTI with the addition of hydrazine.
(2.9 ppb) (Figure S5 in the Supporting Information) which was
lower enough than that of the TLV (10 ppb) recommended by the
EPA and WHO.
In a UVꢀvis absorption spectrum, the solution of BTI showed
dual absorption bands at 306 and 313 nm which may be attributed
4
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To confirm that the fluorescence sensing response of the probe 50 fluorescent compound 2 for a fluorescence turnꢀon response
to hydrazine is indeed due to the conversion of probe BTI to
(Scheme 2).
compound 2, the reaction product of probe BTI with hydrazine
was isolated by column chromatography. The H NMR spectrum
O
1
NH-NH2
O
O
NH2
O
5
of the isolated product is essentially identical with that of the
standard compound 2 (Figure S6 in the Supporting Information),
in good agreement with the formation of compound 2. On the
basis of these experiments and reported literatures,36ꢀ38 we
speculate that the carbonyl position of phthalimide in the BTI
N
N
NH2-NH2
NH
NH
N
N
S
O
NH
N
S
O
N
N
2
S
3
BTI
55
I
10 was selected as the reaction site and the proposed reaction
mechanism of BTI with hydrazine is illustrated in Scheme 2
involving two steps. At first the nucleophilic additionꢀelimination
to the carbonyl group at the phthalimide in the BTI resulted in the
intermediate I and then the second nucleophilic additionꢀ
15 elimination to the another carbonyl group by –NH2 in I resulted
amide ring formation that leads to phthalhydrazide and release
Scheme 2. Proposed hydrazinolysis mechanism of sensor BTI to
hydrazine.
In order to check the practical utility of BTI to detect hydrazine
selectively even in the presence of common anions and cations,
60 redox molecules and amines competitive analyte titrations were
carried out. The BTI fluorescent probe displayed a large
fluorescence turnꢀon response to hydrazine (Figure 3). By
contrast, representative species such as F−, Cl−, Br−, I−, CN−,
the aminobenzthiadiazole (2), which carry out
a unique
chromogenic response. To confirm the validity of the proposed
sensing mechanism, a solution of probe BTI was analyzed by 1H
20 NMR in the absence and presence of hydrazine, as displayed in
Figure 4.
PO43−, NO3− , S2− and SO4 (as their sodium salts), and amines
2−
65 (ethylenediamine, 1,3ꢀdiaminopropane, oꢀphenylenediamine,
ammonia, hydroxylamine, cysteine, homocysteine, urea and
thiourea) exhibited almost no changes in emission behaviour.
Meanwhile, the commonly encountered cations [Cu2+, Hg2+, Zn2+,
Cd2+, Mg2+, Fe3+, Al3+ and Ag+ (as their chloride salts)] did not
70 cause any change to the fluorescence of probe BTI (Figure S8 in
the Supporting Information).
Hg
NH2
O
H d
H f
Hb, Hd, He
He
He
NH
Ha
Hb
N
N
NH
H f
S
H g
Hd
O
H c
Hc
Hb
H a
3
2
Hf
H e
H d
25
Hc, Hb
Ha
Hc
O
a)
Hd
N
N
H a
N
S
He
He
O
Hd
BTI
75
1
Figure 4. Partial H NMR (400 MHz) spectra of BTI only and
30 [BTI + N2H4]. [BTI] = 1 x 10ꢀ3 M, [Hydrazine] = 1 x 10ꢀ2 M in
D2O/DMSOꢀd6 (4:6, v/v).
b)
B T I
2
3
4
5
1
1
8
9
6
6
7
10
After hydrazine was added, the protons Ha and Hc of probe BTI
moved up field from 8.305, 7.939 to 6.597 and 7.143 ppm
respectively, which was almost identical to that of
35 aminobenzthiadiazole (2) (Figure S6 in the Supporting
Information). A new peak at 6.164 ppm also appeared, assignable
to the corresponding –NH2 protons (Hg) of 2. In addition, the
characteristic NH proton resonances of the reaction product
phthalhydrazide was also clearly observed at 8.028 ppm (Figure
40 4) indicating the hydrazinolysis of phthalimide moiety. To further
understand the mechanism of probe BTI with hydrazine, LCMS
was used to test the solutions containing BTI and 2 equiv.
hydrazine, and the peak at 163.2 instead of 282.1 proved that the
benzthiadiazole group had been removed and generation of new
45 peak at 169.3 corresponds to aminobenzthiadiazole (2) (Figure S7
in the Supporting Information). Thus, the extensive studies of
NMR, mass spectrometry, absorption, emission, and excitation
spectroscopy corroborate that indeed, as designed, nonfluorescent
probe BTI was transformed by hydrazine to afford strongly
80
B T I
2
3
4
5
10
7
8
9
85 Figure 5. (a) Relative fluorescence responses of sensor BTI (c =
1 x 10ꢀ6 MLꢀ1) in H2O–DMSO (4:6, v/v) solution (10 mM HEPES
buffer, pH 7.4) to hydrazine (1.5 equivalents) and other various
relevant analytes (50 equivalents each): (1) blank; (2) hydrazine;
(3) F−; (4) Cl−; (5) Br−; (6) I−; (7) CN−; (8) PO43−; (9) NO3− ; (10)
90 S2−; (11) SO42−; (12) Cu2+; (13) Hg2+; (14) Zn2+; (15) Cd2+; (16)
Mg2+; (17) Fe3+; (18) Al3+; (19) Ag+; (20) mixture [BTI+ analytes
(tested anions & cations)+N2H4] and (b) The photograph of
visible color (top) under ambient light and visual fluorescence
color (bottom) changes of sensor BTI (c = 1 x 10ꢀ6 MLꢀ1) with
95 various relevant analytes in H2O–DMSO (4:6, v/v) solution (10
mM HEPES buffer, pH 7.4) under a hand–held UV lamp (366
nm): BTI only; (1) ethylenediamine; (2) 1,3ꢀdiaminopropane; (3)
This journal is © The Royal Society of Chemistry [year]
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fb
Molecules
Electronic
Transition
Experimentally
obtained
Theoretically
obtained
(λmax
oꢀphenylenediamine; (4) ammonia; (5) hydrazine; (6)
hydroxylamine; (7) cysteine; (8) homocysteine; (9) urea; (10)
thiourea.
(λmax
)
)
S0 → S2 313 nm
336.87 nm 0.3052
BTI
5
However the absorption and fluorescence titration carried out
with all the other common cations and anions as well as redox
anions showed no significant change, indicating their
noninteractive nature with BTI (Figure 2b, 5a). Even the different
amines do not react to BTI at room temperature (Figure 3b, 5b).
S0 → S1 395 nm
S0 → S1 315 nm
466.37 nm 0.0571
290.97 nm 0.0647
2
3
55 In addition, we also performed timeꢀdependent density function
theory (TDDFT) calculations for the reactant as well as both the
product also. The vertical transitions i.e, the calculated λmax,
main orbital transition, and oscillator strength (f) are listed in
Table S3 and S4 (Supporting Information). In the case of the BTI
60 probe, TDDFT calculations provided absorption band at ~336 nm
belonging to the S0→S2 (f = 0.3052) energy state. This value is
consistent with the absorbance band at 313 nm (ε= 3.78 × 105 Mꢀ1
cmꢀ1) obtained experimentally. Furthermore, the energy gap
between the HOMO and LUMO of 2 was smaller than that of
65 probe BTI, in good agreement with the apparent red shift (~395
nm) in the absorption observed upon the treatment of probe BTI
with hydrazine. The calculated band at ~466 nm of 2 is assigned
to the vertical major transition of HOMO → LUMO, S0→S1
(~98.80%) that results from an n → π* transition within the
70 aminoꢀbenzothiadiazole moiety of 2, which mainly corresponds
to the experimentally observed absorbance band at 395nm (Table
1).
10 The results indicate that the probe BTI has high selectivity for
hydrazine over other species. This may be attributed to the
unique chemical reaction between phthalimide and hydrazine.
Fluorescence spectra were also recorded for the titration of probe
BTI against hydrazine in the presence of 50 equiv of common
15 anions and cations , redox anions and amines. None of these
analyte significantly affect the emission intensity of BTI upon the
addition of hydrazine, and the titration profile is similar to that
obtained for simple hydrazine titration (Figure 3b, 5a). Therefore,
it can be concluded that probe BTI selectively reacts hydrazine
20 even in the presence of other analytes.
The time course of the fluorescence intensity of the probe BTI
(c = 1 x 10ꢀ6 MLꢀ1) in the absence or presence of hydrazine (1.5
equiv.) in H2O–DMSO (4:6, v/v) solution (10 mM HEPES buffer,
pH 7.4) is displayed in Figure S9 in the Supporting Information.
25 The free probe BTI exhibited no noticeable changes in the
emission intensity at 498 nm. However, upon introduction of
hydrazine, a significant enhancement in the emission intensity
was observed within minutes, and the emission intensity
essentially reached the maximum in 20 minutes. To be useful in
30 biological applications, it is necessary for a probe to function
over a suitable range of pH, in particular at physiological pH. So,
the effect of pH on the fluorescence response of BTI to hydrazine
was investigated. As shown in Figure S10 in the Supporting
Information in the absence of hydrazine, almost no change in
35 fluorescence intensity was observed in the free chemosensor over
a wide pH range of 1.0–11.0, indicating that the free chemosensor
was stable in the wide pH range. Therefore, considering the
environmental and biological applications, all studies were
carried out at the physiologically relevant pH of 7.4 for the
40 detection of hydrazine.
To be useful in practical applications, we further tested whether
probe BTI could be applied for the detection of gas state
75 hydrazine. To make the detection experiments easy to perform
and practical, silica gel TLC plates were used. Prior to detection,
silica gel TLC plates (silica layer of thickness 0.2 mm on
aluminium foil) were prepared by immersing the TLC plates into
a CHCl3 solution of probe BTI (c = 1 x 10ꢀ3 MLꢀ1) and then dried.
80 The probeꢀloaded TLC plates were covered on the top of jars that
contained different hydrazine solution concentrations (blank,
0.01%, 0.1%, 0.5%, 1%, 5%, 10%, 20%, 25%, 30% and 40% in
water) for 15 min at room temperature before it was ready to
observe. As shown in Figure 6, the change in the color of the
85 fluorescence from colorless to green was observed using a handꢀ
held UV lamp with excitation at 366 nm.
To get insight into the optical response of probe BTI to
hydrazine, probe BTI and the corresponding product after
reaction with hydrazine 2 and 3 were examined by density
function theory (DFT) and timeꢀdependent density function
90
45 theory (TDDFT) calculations using
a TDDFT//B3LYP/6ꢀ
31+G(d,p) + solv(SMD) level of the Guassian 09 program.40
Geometries have been optimized in presence of solvent water.
Solvent effects were incorporated using SMD solvent model. The
optimized geometries and calculated electron distributions in the
50 frontier molecular orbitals of BTI, 2 and 3 are shown in Figure
S12 in the Supporting Information.
95 Figure 6. Fluorescence color changes of probe BTI (c = 1 x 10ꢀ3
MLꢀ1) coated silica gel TLC plates (silica layer of thickness 0.2
mm on aluminium foil) after exposure to different concentrations
of hydrazine aqueous solution. The Fluorescence color changes
were collected using a hand–held UV lamp (366 nm).
Table 1. Comparison of the experimental and theoretical TDDFT
data for BTI, 2 and 3 in solvent phase.
100
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The gas state hydrazine detection limit of probe BTI
Figure 8. Fluorescence detection of hydrazine in distilled water
and tap water by BTI. [BTI] = 1.0 × 10−6 MLꢀ1, [Hydrazine] =
from 0 to 1.0 × 10−5 MLꢀ1 in H2O–DMSO (4:6, v/v) solution (10
mM HEPES buffer, pH 7.4) [λexc = 313 nm].
concentration is as low as 0.1%, which is considerably more
sensitive than the recently developed hydrazine probes.29ꢀ30 It is
noteworthy that the probe has high potential applications in
hydrazine detection. Hydrazine in gaseous form often threatens
human life, so our designed probe has more application potential.
5
50 To further demonstrate the application potential of probe BTI in
living cells, the probe was applied in Vero cells for fluorescence
imaging of hydrazine. Prior to investigating the suitability of the
probe BTI for imaging hydrazine in living cells, it is necessary to
evaluate its cytotoxicity. The standard MTT assays suggest that
We further tested whether probe BTI could be applied for the
detection of hydrazine in solution. To make the detection
experiments easy to perform and practical a TLC plate was used.
10 Prior to detection, a silica gel TLC plate (silica layer of thickness 55 the probe BTI does not exert any adverse effect on cell viability
0.2 mm on aluminium foil) was firstly immersed into H2O–
DMSO (4:6, v/v) solution (10 mM HEPES buffer, pH 7.4)
solution of BTI (c = 1 x 10ꢀ3 MLꢀ1) and dried, then the probeꢀ
loaded TLC plate was sink into a beaker containing hydrazine
(Figure S13 in the Supporting Information). Now the stage was
set for cell imaging of hydrazine. The living cells were treated
with the probe BTI in the absence or presence of hydrazine. Vero
0
cells incubated with BTI (10 µM) for 20 min at 37 C in PBS
15 solution for 1.0 min at r.t. before it was ready to observe. As 60 buffer with 0.5%DMSO showed nonfluorescent as shown in
shown in Figure 7, the change in the color of the fluorescence
from dark to green was observed using a handꢀheld UV lamp
with excitation at 366 nm.
Figure 10. By contrast, cells preꢀloaded with the probe BTI and
further incubated with hydrazine for further 10 min displayed
green fluorescence (Figure 9) inside Vero cells, as observed
earlier in solution studies. These findings open up the avenue for
65 future in vivo biomedical applications of the sensor. Thus, BTI is
cell membrane permeable and capable of fluorescence imaging of
hydrazine in the living cells.
(a)
(a)
(b)
(b)
20
N2 H4
A
B
Figure 7. (a) Naked eye detection under ambient lighting
70
conditions and (b) fluorescence color changes visualized on TLC
25 plate strips of sensor BTI (c = 1 x 10ꢀ3 MLꢀ1) in the presence of
hydrazine (c =1 × 10−4 M) in H2O–DMSO (4:6, v/v) solution (10
mM HEPES buffer, pH 7.4).
D
C
We also explored opportunities for probe BTI to analyze
hydrazine in aqueous solution for practical applications. Because
30 hydrazine has carcinogenic properties, and has been widely used
in a variety of industrial processes, hydrazine detection in
aqueous samples is of interest. Prior to living cell imaging, probe
BTI was used to detect hydrazine in tap water and distilled water.
An aliquot of hydrazine was added to water and the recoveries
35 obtained by BTI signals were compared in tap water and distilled
water (Figure 8). The analysis of hydrazine in both solutions
agreed well at hydrazine concentrations up to 10 ꢁM. The results
show that probe BTI can detect hydrazine in real water samples
quantitatively.
75 Figure 9. Confocal Fluorescence microscopic images in Vero 76
cells (Vero 76, ATCC No CRLꢀ1587): (A) bright field image of
the cells, pretreated with probe BTI, (B) only probe BTI at 1.0 x
10ꢀ6 M concentration, (C) bright field image of the cells treated
with N2H4 at concentration 1.0 x 10ꢀ5 M, (D) cells treated with
80 probe BTI at concentration 1.0 x 10ꢀ6 M, N2H4 at 1.0 x 10ꢀ5 M,
and switchꢀon fluorescence signal is detected. All images were
acquired with a 40x confocal objective lens. λex = 405 nm, λem
513 nm.
=
40
Conclusion
85 In this paper, we report a new fluorescent probe BTI for selective
detection of hydrazine both in gasꢀphase and aqueous solution
based on Gabriel’s primary amine synthesis. The probe which is
equipped with
a designed phthalimide framework could
selectively react with hydrazine, resulting in an OFF–ON
90 fluorescence signal change at 498 nm accompanied by a color
change from colorless to green at room temperature. The
selectivity and sensitivity was demonstrated on the basis of
45
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5
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a
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We thank the DSTꢀNew Delhi [Project file no. SR/S1/OCꢀ
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Graphical Abstract
A highly sensitive fluorescent probe for detection of
hydrazine in gas and solution phase based on the Gabriel
mechanism and its bioimaging
Rajkishor Maji, Ajit Kumar Mahapatra, Kalipada Maiti, Sanchita Mondal, Syed Samim Ali,
Prithidipa Sahoo, Sukhendu Mandal, Md Raihan Uddin, Shyamaprosad Goswami, Ching Kheng
Quah and Hoong-Kun Fun
NH2NH2
Fluorescence
Fluorescence
ON
OFF
N2H4
Application
Detection of gas-state hydrazine
Cell image study