A dual functional MOF-based fluorescent sensor for intracellular phosphate and extracellular 4-nitrobenzaldehyde

Article abstract of DOI:10.1039/c8dt03964j

Herein, we present the successful synthesis, characterization and sensing application of a hydrazine functionalized Zr(iv)-based UiO-66 metal-organic framework (MOF) called Zr-UiO-66-N₂H₃ (1). The guest-free material 1′ shows selecti

Full text of DOI:10.1039/c8dt03964j

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A dual functional MOF-based fluorescent sensor
for intracellular phosphate and extracellular
Cite this: DOI: 10.1039/c8dt03964j
4-nitrobenzaldehyde†
a
Aniruddha Das,a Sourik Das,a Vishal Trivedib and Shyam Biswas
*
Herein, we present the successful synthesis, characterization and sensing application of a hydrazine func-
tionalized Zr(IV)-based UiO-66 metal–organic framework (MOF) called Zr-UiO-66-N2H3 (1). The guest-
free material 1’ shows selective and sensitive fluorimetric detection (turn-on mechanism) of phosphate
(PO43−) anions in HEPES buffer (10 mM, pH = 7.4) and aqueous medium. It can also fluorimetrically detect
(turn-off mechanism) 4-nitrobenzaldehyde (4-NB) in a HEPES : DMSO (9 : 1, v/v) medium with high
selectivity and sensitivity. The selectivity for both analytes is retained in the presence of other potentially
3−
3−
competitive analytes. The detection limit for PO4 ions is 0.196 µM, which is far below the PO4 level
present in the aqueous environment. MOF 1’ can detect intracellular phosphate and it also has the
capacity to exhibit differences in the intracellular phosphate level. Furthermore, the probe is capable of
Received 2nd October 2018,
Accepted 11th December 2018
3−
sensing PO4 ions in real samples such as tap water, lake water, human urine and human blood serum.
DOI: 10.1039/c8dt03964j
The sensitivity of the probe for sensing 4-NB is very high (detection limit = 4.7 μM). The possible mecha-
nisms for sensing PO43− ions and 4-NB have been explored in detail by experimental techniques.
rsc.li/dalton
On the other hand, chloramphenicol, which is extensively
used as a first-line therapy for the treatment of typhoid fever
Introduction
Phosphate (PO43−), being an inorganic anion, is a basic frag- patients and most commonly in eye drops (mainly in develop-
ment of nucleotides and plays very vital roles in signal trans- ing countries), gets decomposed in the presence of UV-A radi-
duction and energy storage. The other important roles of this ation of sunlight to produce various decomposition products
anion in biological systems include bone mineralization, such as 4-nitrobenzaldehyde (4-NB), 4-nitrobenzoic acid and
muscle function, membrane robustness, cellular signaling, etc. 4-nitrosobenzoic acid.5 The toxicities of all the three photo-
3−
The maximum permissible level of PO4 ions varies from products towards bone-marrow cells were studied, which con-
0.0143 to 0.143 mM for waste water and it is 0.32 µM for river cluded that 4-NB, 4-nitrobenzoic acid and 4-nitrosobenzoic
3−
water.1 Excessive amounts of PO4 ions in food or water are acid were 20, 6 and 6 times more toxic than chloramphenicol,
often responsible for several digestive problems.2–4 Because of respectively.6 Moreover, the use of this drug leads to an
their detrimental as well as favorable roles, the development of increased amount of methemoglobin (Met-Hb) in blood which
3−
a practical method for the aqueous-phase detection of PO4
is the cause of oxidative stress. Therefore, the in vitro sensing
ions in an extremely selective and sensitive way will be ben- of 4-NB in a cell or in the fluid secreted from eyes being
eficial for environmental protection as well as for human exposed to sunlight after the application of the eye drop is
health.
highly required, since it might be a prior indication of fatal
aplastic anemia disease.7
Due to the unique structures and fascinating properties
such as tunable and extraordinary porosity, high physico-
chemical and mechanical stability and outstanding catalytic
activity, metal–organic frameworks (MOFs) have received exten-
sive research interest in recent years.8–10 Besides gas storage,
chemical catalysis,11,12 chemical separation,13,14 drug deliv-
ery,15,16 and enzymatic catalysis,17–20 MOFs have also been
considered as one of the leading fluorescent sensor materials.
Fluorescent MOFs have been used for the detection of a variety
of organic and inorganic molecules, radicals, ions, etc.21–23
aDepartment of Chemistry, Indian Institute of Technology Guwahati, 781039 Assam,
India. E-mail: sbiswas@iitg.ernet.in; Fax: +91-3612582349; Tel: +91-3612583309
bMalaria Research Group, Department of Biosciences and Bioengineering, Indian
Institute of Technology Guwahati, 781039 Assam, India
†Electronic supplementary information (ESI) available: Synthesis of ligands,
mass spectra, NMR spectra, IR spectra, XRPD patterns, TG curves, N2 sorption
isotherms, fluorescence spectra, FE-SEM images, life-time decay profiles, table
containing unit cell parameters of compound
values. See DOI: 10.1039/c8dt03964j
1 and excited-state life-time
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The choice of appropriate functional groups in the framework
ligands is very crucial for the exhibition of sensing properties,
since specific functional groups can act as binding sites for
Experimental
Materials and general methods
particular analytes, allowing their detection through fluo- The 2-hydrazinyl-1,4-benzenedicarboxylic acid (H2BDC-N2H3)
rescence spectroscopy. ligand was synthesized by following the previously reported
MOFs having high physicochemical stability (air, water, procedure33 for 2-hydrazinyl-4-(methoxycarbonyl) benzoic acid.
acid, base, thermal, etc.) are highly needed for their practical The synthesis and characterization (Fig. S1–S3†) of the ligand
applications. The Zr(IV)-based MOF namely UiO-66 (UiO = are presented in the ESI.† All the chemicals used in this work
University of Oslo) has received much attention in the past few were commercially available and they were used without any
years due to its high thermal and chemical stability as well as further purification. For fluorescence sensing experiments
significant microporosity.24–28 The 3D cubic framework of this Milli-Q water was used as a medium. Fourier transform infra-
MOF is constructed from [Zr6O4(OH)4]12+ building units, red spectroscopy was performed in the region 400–4000 cm−1
which are interconnected by the carboxylate groups of 1,4-ben- with a PerkinElmer Spectrum Two FT-IR spectrometer. The fol-
zenedicarboxylate (BDC) ligands.29 In compound 1, the BDC lowing indications were used to indicate the corresponding
ligand molecules are replaced by the 2-hydrazinyl-1,4-benzene- absorption bands: very strong (vs), strong (s), medium (m),
dicarboxylate (BDC-N2H3) ligands. The guest-free form of 1 weak (w), shoulder (sh) and broad (br). Thermogravimetric
3−
(termed 1′) can sense PO4 ions in water and HEPES buffer analyses (TGA) were carried out using a SDT Q600 thermo-
(10 mM, pH = 7.4). It can also detect 4-NB in the HEPES gravimetric analyzer in the temperature range 25–700 °C under
(10 mM, pH = 7.4)/DMSO (v/v, 9 : 1) medium. The detection an argon atmosphere at a heating rate of 10 °C min−1. A Bruker
processes for both analytes occur with high selectivity, which D2 Phaser X-ray diffractometer (30 kV, 10 mA) using Cu-Kα (λ =
is preserved in the presence of intrusive analytes (Scheme 1). 1.5406 Å) radiation was used to obtain the X-ray powder diffr-
Herein, we report the highly selective and sensitive fluo- action (XRPD) patterns. An Edinburgh Instrument Life-Spec II
3−
rescence sensing properties of 1′ for PO4 ions and 4-NB via equipment was used to measure the fluorescence lifetimes
turn-on and turn-off mechanisms, respectively.
(TRPL) by employing the time-correlated single-photon count-
1
In addition to its high physicochemical stability, a Zr-based ing (TCSPC) procedure. The solution H and 13C NMR spectra
MOF was chosen for the sensing of PO43− ions due to the high were recorded on a Bruker AM 600 spectrometer. Nitrogen
3−
binding affinity of Zr(IV) ions with PO4 ions. There is already sorption isotherms were recorded on
a Quantachrome
3−
a report in the literature on PO4 sensing by a Zr-based Autosorb iQMP gas sorption analyzer. Fluorescence emission
MOF.30 Moreover, the Zr(IV) ions having a d0 electronic con- studies were performed using
HORIBA JOBIN YVON
a
figuration are electronically inert. Hence, there is good elec- Fluoromax-4 spectrofluorometer. The compound was heated at
tronic communication between the Zr(IV) ions and BDC-N2H3 120 °C for 24 h before the sorption experiments.
ligands.31,32 As a result, 1′ showed a high fluorescence
Synthesis of [Zr6O4(OH)4(BDC-N2H3)6]·3.5H2O·3DMF
(Zr-UiO-66-N2H3, 1)
intensity.
A mixture of ZrOCl2·8H2O (65 mg, 0.20 mmol), H2BDC-N2H3
ligand (40 mg, 0.20 mmol) and benzoic acid (746 mg,
6.0 mmol) in 3 mL of N,N-dimethylformamide (DMF) was
sealed in a glass tube and heated using a block heater at
120 °C for 24 h. The light yellow precipitate was collected by
vacuum filtration and washed with acetone (2 × 3 mL). The
material was dried at 60 °C for 4 h in a conventional oven. The
yield was 45 mg (0.02 mmol, 60%) based on the Zr salt. FT-IR
(KBr, cm−1): 3420 (br), 2916 (w), 2853 (w), 1658 (s), 1627 (vs),
1422 (s), 1418 (vs), 1381 (m), 1188 (m), 1100 (w), 1028 (m), 900
(m), 880 (sh), 845 (s), 782 (vs), 711 (sh), 663 (vs), 558 (w),
495 (s).
Activation of the compound
To obtain the activated form, 50 mg of the as-synthesized com-
pound was stirred for 24 h in 30 mL of methanol in a round-
bottom flask at room temperature, during which fresh metha-
nol was added discarding the initially added methanol at a
time interval of 12 h. After that the compound was filtered and
the solid powder was dried at 80 °C in a conventional oven for
Scheme 1 Schematic representation displaying the sensing properties
of 1’ towards PO4 ions in aqueous medium and 4-NB in the HEPES/
3−
1 h. After that, the compound was heated at 120 °C under
DMSO (v/v, 9 : 1) medium through fluorescence turn-on and turn-off
mechanisms, respectively.
vacuum for 24 h to obtain material 1′.
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Preparation of the suspensions of 1′ for fluorescence sensing
experiments
at 5000 rpm for 5 min and then filtered with Amicon®Ultra-4
Centrifugal Filter Units (30 kDa). The filtrate was collected
which was diluted 100 times with HEPES buffer (10 mM, pH =
7.4). The resulting liquid was used for the sensing experiment.
These samples were distributed in twenty four glass vials so
that four vials contain samples of one type. After that, a known
amount of Na3PO4 was spiked into every vial so that the final
concentration of PO43− ions in four different glass vials having
samples of one kind become 0, 10, 20 and 30 µM. Then, each
To obtain stable suspensions for the luminescence detection
experiments, 2 mg of 1′ was taken in three separate 5 mL glass
vials. Then, 3 mL of water, HEPES (10 mM, pH = 7.4) (the first
3−
two for PO4 sensing) or a HEPES (10 mM, pH = 7.4)/DMSO
(v/v = 9 : 1) mixture (for 4-NB sensing) was separately added to
these vials. These mixtures were allowed to sonicate for 1 h
and subsequently kept for 24 h under ambient conditions.
3−
PO4 spiked sample (100 μL) was added to the aqueous sus-
pension (2900 μL) of 1′. We recorded the fluorescence spec-
trum after 100 min of addition.
Fluorescence sensing experiments
3−
In order to perform the sensing experiment for PO4 ions,
100 μL of the suspension of 1′ from the stock suspension was
placed in a quartz cuvette and 2900 μL of water or HEPES
buffer was added to it in order to make a suspension having a
Results and discussion
Preparation and activation procedure
3−
total volume of 3 mL. Then, a solution of PO4 ions was
added to it up to 400 µL with an incremental volume of 50 µL
and the emission spectra were recorded after every 5 or 10 min
until the fluorescence intensity reached the saturation point.
For the sensing of 4-NB, 100 μL of the suspension of 1′ from
the stock suspension was taken in a quartz cuvette and
2900 μL of the HEPES/DMSO (v/v = 9 : 1) mixture was added to
it in order to make a suspension having a total volume of
3 mL. Then, a solution of 4-NB in DMSO was added to it up to
400 µL with an incremental volume of 50 µL and the fluo-
rescence spectra were recorded until the saturation point for
the fluorescence intensity was attained. For all these cases, the
suspension of 1′ was excited at 360 nm and the fluorescence
spectra were recorded in the range of 385–585 nm. The fold
Before determining the optimized synthesis conditions for 1,
several possible reactions were carried out with the
H2BDC-N2H3 ligand using three Zr(IV) salts (ZrCl4,
ZrOCl2·8H2O) in the presence of two different amide solvents
(N,N-dimethylformamide, N,N-dimethylacetamide) and four
different modulators (acetic acid, formic acid, benzoic acid
and trifluoroacetic acid)37 at different temperatures. Highly
crystalline compound 1 was obtained when a mixture of
ZrOCl2·8H2O, H2BDC-N2H3 ligand and benzoic acid was
placed in DMF in a sealed glass tube and heated at 120 °C for
24 h using a block heater.
In order to remove the guest molecules trapped inside the
pores of 1, an activation procedure was performed which
involves a solvent-exchange step followed by a heating step
under high vacuum. In the solvent-exchange step, 100 mg of 1
was suspended in 30 mL of methanol inside a 50 mL round-
bottom flask and stirred for 24 h at room temperature. Then, it
was filtered and dried at 60 °C in a conventional oven.
Furthermore, it was activated at 120 °C under vacuum for 24 h.
3−
increment for the PO4 sensing via the turn-on mechanism
was calculated using the formula: fold increment = (I/I0 − 1).
The quenching efficiency (η) for the 4-NB sensing was calcu-
lated using the familiar equation: η = (1 − I/I0) × 100%, where
I0 is the initial fluorescence intensity of the suspension of 1′
and I is the fluorescence intensity after the addition of the
analyte.
Infrared spectroscopy
Cell culture and intracellular PO43− sensing
In the FT-IR spectra (Fig. S4, ESI†) of the as-synthesized 1 and
activated 1′, the strong absorption bands at around 1570 and
1400 cm−1 can be attributed to the asymmetric and symmetric
carboxylate stretching vibrations of the coordinated BDC-N2H3
ligand molecules, respectively.38 In the IR spectrum of the as-
synthesized sample, the strong absorption band at 1658 cm−1
can be attributed to the carbonyl stretching vibration of the
occluded DMF molecules in the framework structure.39 This
absorption band is completely absent in the FT-IR spectrum of
1′, which confirms that the thermally activated compound is
devoid of any guest DMF molecule inside the pores.
The macrophage J774A.1, cervical cancer HeLa and glioblas-
toma U87MG cells were cultured in DMEM:F12 media contain-
ing 10% FBS and 1% antibiotic cocktails as described
earlier.34–36 The cells were loaded with probe 1′ (0.5 mg mL−1
)
for 6 h in complete medium. The cells were observed in the
bright field and the blue channel (λex = 390 nm, λem = 430 nm)
using the Cytell cell imaging system (GE Healthcare) and
images were captured from randomly selected fields.
PO43− sensing in real samples
3−
We analyzed PO4 ions in six different types of real samples:
XRPD analysis
tap and lake water (collected locally), urine 1 and 2 (collected
from two different human volunteers), and blood serum 1 and The experimental XRPD patterns (Fig. 1) of 1 and 1′ match
serum 2 (collected from two different human volunteers). nicely with the simulated XPRD pattern of the previously
Human urine samples were kept at 4 °C overnight and then reported UiO-66 compound.40,41 By indexing the XRPD pattern
the supernatant liquid was collected which was used for the of 1, its unit cell parameters were deduced. The observed
sensing experiment. Human serum samples were centrifuged lattice parameters (Table S1, ESI†) are very close to the
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and the inorganic building units together make the framework
three-dimensional via the interconnection with each other.
Thermal stability
For investigating the thermal stability of 1 and 1′, thermo-
gravimetric (TG) analyses were performed under an argon atmo-
sphere in the temperature range of 25–700 °C. According to
the TG analyses, 1 is thermally stable up to 400 °C under the
air atmosphere.
In the TG curve (Fig. S7, ESI†) of 1, the first loss of 2.6 wt%
in the temperature range of 25–120 °C can be attributed to the
elimination of 3.5 water molecules per formula unit (cald:
2.5 wt%). The second weight loss of 12.2 wt% in the tempera-
ture range of 120–400 °C can be assigned to the removal of 3
occluded DMF molecules per formula unit (cald: 11.8 wt%).
After 400 °C, the decomposition of the compound starts to
occur owing to the loss of organic ligands from the framework
structure of 1. In the TG trace of 1′, the one weight loss in the
low temperature range can be assigned to the removal of
adsorbed water molecules from moisture. It is worth noting
that the thermal stability of our compound 1′ is lower than the
existing, parent and functionalized Zr-based UiO-66 MOF
materials.44–46
Fig. 1 XRPD patterns of the (a) simulated Zr-UiO-66, (b) as-synthesized
1 and (c) activated 1’.
reported, un-functionalized Zr-UiO-66 compound. In addition,
a Pawley fit (Fig. S5, ESI†) was carried out, which displayed
very good similarity between the experimental and calculated
XRPD patterns of 1. All these results suggest that the presented
compound has the same framework topology as the
UiO-66 material.40
The noticeable similarity between the XRPD patterns of the
as-synthesized and thermally activated samples of 1 indicates
that the compound retained its structural robustness after the
thermal activation process. We also obtained the XRPD pat-
terns of 1′ after the fluorescence sensing (for phosphate and
4-NB) measurements. The XRPD patterns displayed in Fig. S6
(ESI†) disclose that the crystallinity and hence the structural
integrity of the material remain unaltered after the fluo-
rescence sensing measurements. These results suggest that 1′
is a very effective potential material for real-world sensing
applications.
Chemical stability
The chemical stability of 1′ was investigated by stirring the
samples in water, acetic acid and 1M HCl solutions at room
temperature for 12 h. After that, the samples were collected by
filtration and the crystallinity of the filtered materials was
checked by XRPD analysis. As shown in Fig. S6, ESI†, the com-
pound retained its crystallinity (and thus structural robust-
ness) after treatment with water, acetic acid and 1M HCl solu-
tions. Hence, 1′ exhibited remarkable chemical stability which
is comparable to that of the previously reported parent and
other functionalized UiO-66 materials.47
N2 sorption analysis
The N2 sorption experiments were carried out for the determi-
nation of the specific surface area and the micropore volume
of 1′. From Fig. S8 (ESI†), it is obvious that the N2 adsorption
isotherm follows type-I behavior, which is characteristic of
Structure description
As confirmed by the XRPD experiments (Fig. 1), the hydrazine- microporous materials. The BET surface area and micropore
functionalized Zr(IV)-based UiO-66 material (1) shows the same volume of 1′ were estimated to be 818 m2 g−1 and 0.47 cm3 g−1
framework topology as the un-functionalized UiO-66 material. (at p/p0 = 0.5), respectively. This value of the BET surface area
Lillerud and co-workers have previously described the frame- is comparable to the known, functionalized UiO-66 MOF
work structure of the UiO-66 material.42 The UiO-66 framework materials.48–54
contains hexanuclear [Zr6O4(OH)4]12+ bricks as the secondary
We also performed N2 sorption measurements (Fig. S9,
building units which are interlinked by the carboxylates of ESI†) with the compound recovered after the phosphate
twelve 1,4-benzenedicarboxylate (BDC) ligands. In the pre- sensing experiment. As anticipated, the BET surface area and
sented work, BDC-N2H3 plays the same role as the BDC ligand micropore volume (at p/p0 = 0.5) of the phosphate-treated
in the previously reported42,43 UiO-66 framework structure. material were lower than the untreated material and they cor-
This 3D framework contains octahedral as well as tetrahedral responded to 442 m2 g−1 and 0.25 cm3 g−1, respectively.
cages. Each central octahedral cage is connected by eight
Photoluminescence properties of 1′
corner tetrahedral cages via narrow triangular windows. Each
zirconium atom resides in a square anti-prismatic coordi- We studied the solid-state fluorescence properties of both the
nation environment of the framework. The organic connecter free H2BDC-N2H3 ligand and 1′. The free H2BDC-N2H3 ligand
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displayed a weak fluorescence intensity (λmax = 447 nm) and 1′ tion did not result in any considerable increment in the fluo-
exhibited a strong luminescence intensity (λmax = 478 nm) in rescence intensity.
3−
the solid state (Fig. S10, ESI†). The fluorescence emission
The detection performance of 1′ towards PO4 ions was
band of the free ligand arises due to the π–π* electronic tran- evaluated by performing time-dependent sensing experiments.
3−
sition of the aromatic ring.55 The enhanced luminescence Upon the addition of 400 μL of the 2 mM PO4 solution, the
intensity of 1′ as compared to the free ligand can be assigned fluorescence emission spectra of 1′ (in both aqueous and
to the ligand-to-metal charge transfer (LMCT).56,57 The blue HEPES buffer media) were recorded at a regular time interval
shift in the λmax value of 1′ as compared to the free ligand of 10 min until the saturation in the fluorescence emission
might also be assigned to the LMCT transition.58,59
intensity was attained. The addition of the PO4 solution to
3−
We also investigated the luminescence properties of both the suspension of 1′ led to the gradual transformation of the
the free H2BDC-N2H3 ligand and 1′ in the aqueous medium. compound into a turn-on state from its initial turn-off state.
The free ligand showed a strong luminescence intensity (λmax
=
The saturation in the fluorescence emission intensity of 1′
443 nm) and 1′ exhibited a weak luminescence intensity occurred after 90 and 100 min in aqueous (Fig. 3) and HEPES
(λmax = 458 nm) in the aqueous medium (Fig. S10, ESI†). This (Fig. S11, ESI†) buffer media, respectively. Therefore, the com-
difference in the luminescence intensity can be ascribed to the pound is a potential fluorescent turn-on probe for the detec-
3−
interaction between the Zr–O nodes and water molecules. tion of PO4 ions under both aqueous and physiological
When water molecules enter inside the pores of 1′, they can conditions.
3−
form hydrogen bonding with the Zr–O nodes of 1′ and thus
The high selectivity of a sensor towards PO4 ions over
perturb the electron transfer from the ligand to metal (i.e., other potentially competing anions is very crucial for practical
LMCT transition).60 This perturbation reduces the lumine- applications. Hence, we measured the fluorescence turn-on
scence intensity of 1′ in the aqueous medium.
response of 1′ (in both water and 10 mM HEPES buffer)
towards various potentially interfering inorganic anions and
3−
organic molecules containing PO4 ions such as NaCl, NaBr,
Sensing behavior towards PO43− ions
NaF, NaH2PO4, Na2HPO4, Na3P2O7, NaI, NaNO2, NaNO3,
NaOAc, Na2S, Na2SO4, NaHS, NaCN, NaHCO3, NaClO4, NaSCN,
adenosine diphosphate (ADP) and adenosine triphosphate
(ATP). It can be seen from Fig. 4 and Fig. S12–S13 (ESI†) that
In order to determine the potential of UiO-66-N2H3 as a fluoro-
3−
genic sensor for PO4 detection in the aqueous or HEPES
buffer medium, fluorescence titration experiments were per-
formed with the suspension of 1′. The fluorescence emission
(λex = 360 nm) spectra of the suspension of 1′ were recorded in
the range of 385–585 nm upon gradual addition (50 μL for
each addition) of 2 mM PO43− solution. The fluorescence emis-
sion intensities were regulated at 430 nm. As shown in Fig. 2,
the saturation of the fluorescence intensity was observed after
3−
the addition of an inorganic PO4 solution to the suspension
of 1′ causes a dramatic increment in the fluorescence emission
intensity. In the presence of all the other possibly competing
anions, there was almost negligible enhancement in the fluo-
3−
rescence emission intensity as compared to the PO4 anion.
These results suggest that the fluorescent turn-on probe is
highly selective towards PO43− ions over other potentially inter-
fering anions.
3−
the addition of 400 µL of the 2 mM PO4 solution to the
aqueous suspension of 1′. Further addition of the PO4 solu-
3−
Fig. 2 Enhancement of the fluorescence intensity of 1’ (0.87 mM) upon
gradual addition of 400 μL of the 2 mM aqueous solution of PO4 ions
Fig. 3 Enhancement of the fluorescence intensity of 1’ (0.87 mM) with
time upon addition of 400 µL of the 2 mM aqueous solution of PO4
3−
3−
(λex = 360 nm, λem = 430 nm).
ions (λex = 360 nm, λem = 430 nm).
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ation of the initial intensity of 1′ without the analyte and m
corresponds to the slope of the above-stated linear curve.62 The
3−
LOD value of 1′ for PO4
sensing was estimated to be
0.196 µM in the aqueous medium. This LOD value is lower
than that of the UiO-66-NH2 compound, which displayed a
LOD of 1.25 µM.30
3−
The recyclability of the detection ability of 1′ towards PO4
ions was investigated up to two cycles. After the first cycle of
the fluorescence sensing experiment, the dispersed MOF
material was collected by centrifugation, washed with copious
amounts of water and finally dried in a conventional oven at
60 °C for 3 h. The material recovered in this way was employed
in the second cycle. The compound showed an already high
Fig. 4 Change in the fluorescence intensity of the suspension of 1’
(0.87 mM) upon the addition of 400 µL of 2 mM aqueous solutions of
various anions (λex = 360 nm, λem = 430 nm).
3−
fluorescence intensity before the addition of the PO4 solu-
tion in the second cycle and a very less increment in the fluo-
3−
rescence intensity after the addition of PO4 in the same
For practical applications in complicated biological
systems,4,61 the high selectivity of a fluorescent sensor material
towards the target analyte in the presence of other potentially
interfering analytes is highly required. For accomplishment of
this purpose, we carried out competitive fluorescence titration
experiments in which the PO43− solution was added to the sus-
pension of 1′ (in aqueous or HEPES buffer media), which also
contained the potentially competing anions. From Fig. 5 and
Fig. S14–S15 (ESI†), it becomes obvious that the significant
cycle. Thus, 1′ showed a dramatic decrease in the fluorescence
3−
turn-on response towards PO4 detection in the second cycle
(Fig. S17, ESI†). Therefore, the material displayed poor recycl-
3−
ability of its detection capability towards PO4 ions in the
aqueous medium.
Mechanism for PO43− sensing
Several instrumental techniques such as FT-IR spectroscopy,
N2 sorption experiment and XRPD analysis were employed to
3−
fluorescence turn-on response of the compound towards PO4
3−
investigate the probable mechanism of PO4 detection by the
ions is retained even in the presence of other potentially com-
peting anions under both aqueous and physiological con-
ditions. Therefore, the remarkable selectivity of 1′ towards
UiO-66-N2H3 material. After incubation with an aqueous solu-
3−
tion of PO4 ions, the compound showed broad absorption
bands between 900 and 1190 cm−1 in the IR spectrum (Fig. S4,
3−
PO4 ions is retained even when other potentially interfering
ESI†) due to P–O stretching vibrations,63 which confirmed the
anions are present in the solution.
3−
coordination of PO4 ions with the framework of 1′. This
The limit of detection (LOD) of the aqueous suspension of
3−
might indicate the complexation between the PO4 ions and
3−
1′ towards PO4 ions was determined by regulating the fluo-
Zr–O clusters because the P–O bonds would limit their stretch-
ing vibrations, resulting in a decrease of their vibrational fre-
quencies.30 The symmetric stretching vibrations of the carbox-
ylate groups in phosphate-incubated 1′ exhibited a blue shift
(from 1385 cm−1 to 1392 cm−1) as compared to untreated 1′.
rescence emission intensity upon gradual addition of a very
low concentration of the PO43− solution to the suspension of 1′
in the aqueous medium. The plot of the fluorescence emission
intensity of the aqueous suspension of 1′ against the concen-
3−
tration of the PO4
solution resulted in a linear curve
3−
This might provide further evidence that PO4 ions have a
(Fig. S16, ESI†). The LOD value was determined according to
the formula: LOD = 3σ/m, where σ represents the standard devi-
coordination effect with the Zr–O clusters and weaken the
interactions between the BDC-N2H3 ligands and Zr–O clusters,
thus making the carboxylate groups more free and their
vibrations stronger.64 Moreover, the phosphate-treated 1′
showed a BET surface area of 442 m2 g−1 (Fig. S9, ESI†), which
is significantly lower as compared to the untreated sample
(828 m2 g−1). Furthermore, 1′ was treated with phosphate in
different molar ratios with respect to zirconium, and XRPD
measurements were carried with these phosphate-treated
samples. Upon increasing the PO43− : Zr molar ratio, the frame-
work of 1′ gradually collapsed (Fig. S18, ESI†) and conse-
quently the organic linker became free. These observations
indicate that the fluorescence turn-on response of 1′ upon the
3−
addition of the PO4 solution can be attributed to the com-
petitive coordination effect. Upon the gradual addition of the
PO4 solution, the interactions between the attached carboxy-
late groups and the Zr–O clusters become relatively weaker as
Fig. 5 Change in the fluorescence intensity of the suspension of 1’
3−
(0.87 mM) upon the addition of 400 µL of the 2 mM aqueous solution of
3−
PO4 ions in the presence of 400 µL of 2 mM aqueous solutions of
other potentially competing anions (λex = 360 nm, λem = 430 nm).
compared to the un-treated compound. The ligand-to-metal
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3−
Table 1 Detection performance of 1’ for PO4
human urine and human blood serum samples
ions in real water,
charge transfer (LMCT) transition from the ligand released in
this way into the solution might enhance the fluorescence
emission intensity.
Na3PO4
spiked (μM)
Na3PO4
Recovery
(%)
Sample name
Tap water
founda (μM)
Effect of pH for PO43− sensing in HEPES buffer
0
10
20
30
0
5.30 0.75
8.72 0.91
19.62 1.93
29.66 1.18
5.74 0.69
9.18 3.92
20.08 0.15
29.23 0.61
58.21 1.44
9.78 3.27
19.56 1.53
29.52 0.92
63.73 0.59
9.80 1.73
19.68 1.42
29.57 1.21
34.58 0.12
9.73 0.62
19.30 0.72
28.42 0.45
35.98 1.63
9.55 1.98
19.57 1.33
28.36 1.13
NA
87.20
98.10
98.87
NA
91.80
100.40
97.43
NA
97.80
97.80
98.40
NA
98.00
98.40
98.57
NA
97.30
96.50
94.73
NA
95.5
97.85
94.53
3−
We carried out both concentration and time-dependent PO4
sensing experiments at four different pH (pH = 3.4, 5.4, 7.4
and 9.4) values in 10 mM HEPES buffer. The results (Fig. S19–
S23, ESI†) from the concentration-dependent fluorescence
sensing experiments showed that the fold increments (I/I0) in
the fluorescence intensity for 1′ after treatment with 400 μL of
Lake water
10
20
30
0
Human urine 1
10
20
30
0
3−
the 2 mM PO4 solution were 6.7, 26.0, 12.7 and 0.9 at a pH
of 3.4, 5.4, 7.4 and 9.4, respectively. Thus, the fold increment
was the maximum at pH = 5.4 whereas the fold increment was
the minimum at a highly basic pH (pH = 9.4). The fold incre-
ment increased on increasing the solution pH from 3.4 to 5.4
but it decreased at higher pH values (pH = 7.4 and 9.4). At
pH = 3.4, the degree of protonation of the PO43− ions is higher
compared to that at pH = 5.4. Hence, a smaller amount of
Human urine 2
10
20
30
0
10
20
30
0
Human blood serum 1
Human blood serum 2
3−
PO4 ions is available for binding with Zr(IV) ions at pH = 3.4
10
20
30
than at pH = 5.4. As a result, 1′ showed a lower fold increment
at pH = 3.4 compared to that at pH = 5.4. At basic pH, partial
decomposition of the framework of 1′ occurs. Moreover, in a
basic medium, the –NH proton of the hydrazinyl group can be
deprotonated.65 As a result, 1′ showed a high fluorescence
a Na2S found RSD (n = 3).
3−
intensity at basic pH, even before the addition of the PO4
Intracellular PO43− sensing
Before performing the live cell imaging experiment for PO4
solution. Therefore, the fold increment in the fluorescence
intensity decreased with the increment in pH from 7.4 to 9.4.
3−
The saturation times (i.e., time to reach the saturation point ions, we checked the particle size of 1′ by FE-SEM. Fig. S27
in fluorescence intensity after the addition of 400 µL of the (ESI†) reveals that 1′ has an average particle size of less than
3−
2 mM PO4 solution) were 45, 75, 100 and 5 min at a pH of 100 nm. Thus, its particles can easily penetrate through the
3.4, 5.4, 7.4 and 9.4, respectively (Fig. S11 and S24–S26, ESI†). cell membrane.
With increasing pH, the saturation time increased up to pH =
The intrinsic phosphate species (inorganic phosphate and
7.4. At pH = 9.4, 1′ was already in a highly fluorescent state, other phosphorylated derivatives) varies among different cell
3−
even before the addition of the PO4 solution. Therefore, the types and may correlate strongly with metabolism and the
fluorescence intensity of 1′ did not increase significantly with energy status.66,67 To check the potential of 1′ to perform intra-
time after the addition of the PO43− solution at pH = 9.4.
cellular phosphate detection, fluorescence imaging experi-
ments were performed in macrophage J774A.1, cervical cancer
HeLa and glioblastoma U87MG cells. The unlabeled cells were
healthy and they did not exhibit any blue fluorescence (Fig. 6a,
PO43− sensing in real samples
3−
As compound 1′ is highly sensitive towards PO4 ions in g and m). Macrophage J774A.1 cells loaded with the probe
aqueous as well as in the HEPES medium, we decided to (Fig. 6d, J774 panel) were healthy and exhibited blue fluo-
perform the sensing experiments with real biological and rescence (Fig. 6e, J774 panel). The superposition of the phase
environmental samples. For this purpose, we chose a total of and fluorescence indicates that the signal arises from the cells
six samples of three different types (water, human urine and (Fig. 6f, J774 panel, superimposed). HeLa cancer cells loaded
human blood serum). We spiked each of these samples with with the probe showed a distorted cellular morphology but
3−
known concentrations of the PO4 solution. Then, we treated this could be due to the uptake of the suspension of the probe
the MOF probe with these spiked samples and recorded the (Fig. 6g and j, HeLa panel). These cells exhibited intense
fluorescence spectra after 90 min. After the spiked-and-recov- bright blue fluorescence (Fig. 6h and k, HeLa panel, fluo-
3−
ery experiments, the amount of PO4 ions found and the rescence). The superposition of the phase and fluorescence
recovery percentages are summarized in Table 1. In the case of indicates that the signal arises from the cells (Fig. 6i and k,
all the samples, excellent recovery percentages and low RSD HeLa panel, superimposed). The probe loaded glioblastoma
values make probe 1′ a smart candidate for quantifying the U87MG cells displayed strong fluorescence signals (Fig. 6n and
3−
PO4 concentration present in different real and complicated q, HeLa panel, fluorescence), and the superposition of the
biological as well as environmental samples.
phase and fluorescence indicates that the signal stems from
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The compound also exhibited a high fluorescence intensity in
DMSO/HEPES (10 mM, pH = 7.4) buffer (v/v = 9 : 1) (Fig. S28,
ESI†). Furthermore, the compound showed outstanding
selectivity towards 4-NB over other aldehydes in the DMSO/
HEPES buffer (v/v = 9 : 1) medium. Therefore, this solvent
mixture was chosen as the medium for preparing the suspen-
sion of 1′ in order to perform the sensing experiments for
4-NB.
In order to determine the potential of Zr-UiO-66-N2H3 as a
fluorogenic sensor for 4-NB detection, fluorescence titration
experiments were performed with the suspension of 1′ in the
DMSO/HEPES (9 : 1, v/v) mixture, even in the presence of
potentially competitive aldehydes. The fluorescence emission
(λex = 360 nm) spectra of the suspension of 1′ were recorded in
the range of 385–585 nm upon gradual addition (50 μL for
each addition) of the 50 mM 4-NB solution. The fluorescence
emission intensities were regulated at 430 nm. As shown in
Fig. 7, the saturation of the fluorescence intensity was
observed after the addition of 400 µL of the 50 mM 4-NB solu-
tion. Further addition of the 4-NB solution did not result in
any considerable decrease in the fluorescence intensity.
For real-world applications of the sensor material, its high
selectivity for 4-NB over other potentially competing aldehydes
is highly required. Therefore, the fluorescence turn-off
responses of 1′ towards various other aldehydes were recorded
including formaldehyde, benzaldehyde, acetaldehyde, 4-chlor-
obenzaldehyde, valeraldehyde, butyraldehyde, crotonaldehyde,
anisaldehyde, propionaldehyde, 2-nitrobenzaldehyde (2-NB)
and 3-nitrobenzaldehyde (3-NB). As shown in Fig. 8, the
addition of the 4-NB solution to the suspension of 1′ in the
DMSO/HEPES mixture resulted in
a dramatic decrease
Fig. 6 Three different types of cells, macrophage J774A.1 (J774 panel),
cervical cancer HeLa (HeLa panel) and glioblastoma U87MG (U87MG
panel) were either as such (a, b, g, h, m, n) or loaded with probe 1’ (d, j,
p). Probe loaded cells exhibited bright blue fluorescence (e, k, q) and
superposition of the bright field (c, i, o) and fluorescence (f, l, r) indicates
(quenching efficiency = 92%) in the fluorescence emission
intensity. The fluorescence quenching efficiencies of all other
potentially competing aldehydes were relatively less as com-
pared to that of 4-NB (Fig. S29, ESI†). Hence, it can be con-
that the signal arises from the cells associated with the probe (λex
390 nm, λem = 430 nm).
=
the cells (Fig. 6o and r, U87MG panel, superimposed). The
comparison of fluorescence in all three cells indicates that the
fluorescence signal is more in HeLa and U87MG cells com-
pared to the J774A.1 cells. This difference in fluorescence
could be due to the inherent difference in their intracellular
total phosphate level68–70 or because of the differential levels
of stress inside the dissimilar cells owing to probe loading.
Overall, probe 1′ has the potential to detect intracellular phos-
phate and it also has the potential to show difference in the
intracellular phosphate level.
Sensing behaviour towards 4-NB
The fluorescence emission spectra of 1′ (Fig. S28, ESI†) were
recorded in water as well as various organic solvents such as
acetonitrile, methanol, ethanol, toluene, DMF and dimethyl-
sulphoxide (DMSO).71 Among the different solvents tested, 1′
Fig. 7 Quenching of the fluorescence intensity of 1’ (0.87 mM) by
incremental addition of 400 µL of the 50 mM 4-NB solution to a 3 mL
stable suspension of 1’ in HEPES/DMSO (9 : 1, v/v) (λex = 360 nm, λem
=
showed the highest fluorescence emission intensity in DMSO. 430 nm).
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The limit of detection (LOD) of 1′ for 4-NB was evaluated by
regulating the fluorescence emission intensity of the com-
pound upon gradual addition of very low concentrations of the
4-NB solution in the DMSO/HEPES (9 : 1, v/v) mixture. A linear
curve (Fig. S31, ESI†) was achieved when the fluorescence
intensity of 1′ was plotted against the concentration of the
4-NB solution. The LOD value was estimated by using the
formula: LOD = 3σ/m, where σ is the standard deviation of the
initial intensity of 1′ without the analyte and m denotes the
slope of the above-stated linear curve.62 The LOD value of 1′ for
4-NB was found to be 4.7 μM.
For checking the recyclability of the detection capability of
1′ towards 4-NB, fluorescence sensing experiments were
carried out up to four cycles. The compound was collected by
centrifugation after each cycle of the fluorescence sensing
measurement. Then, it was washed with a sufficient amount of
acetone and finally dried in a conventional oven at 70 °C for
30 min. After the first cycle, the fluorescence emission inten-
sity of 1′ decreased significantly. However, there was no con-
siderable decrease in the fluorescence emission intensity after
the subsequent cycles. Therefore, the fluorescence quenching
efficiency of the compound decreased considerably in the
second cycle, although it remained almost unaltered in the
later cycles (Fig. S32, ESI†). Therefore, 1′ exhibited partial
recyclability of its detection performance towards 4-NB.
For determination of the nature of the quenching (static
versus dynamic) process, we analyzed the fluorescence quench-
ing data by using the Stern–Volmer (S–V) equation which is
shown below: towards 4-NB.
Fig. 8 Fluorescence quenching efficiencies of different aldehydes
towards 1’ (0.87 mM) in the DMSO/HEPES (9 : 1, v/v) mixture. The fluo-
rescence spectra were recorded after the addition of 400 μL of 50 mM
aldehyde solutions (λex = 360 nm, λem = 430 nm).
cluded that 1′ is highly selective towards 4-NB over other
potentially intrusive aldehydes in the DMSO/HEPES mixture.
A fluorescent probe must show high selectivity towards the
target analyte in the presence of other possibly competing ana-
lytes, which is highly required for the practical applications of
a fluorescent sensor material in complex biological media.72,73
In order to fulfil this purpose, we carried out competitive fluo-
rescence titration experiments in which the 4-NB solution was
added to the suspension of 1′, which also contained the poss-
ibly competing aldehydes. It is obvious from Fig. 9 and
Fig. S30 (ESI†) that the outstanding fluorescence quenching
efficiency of the compound towards 4-NB was retained even in
the presence of other possibly interfering aldehydes in the
DMSO/HEPES mixture. Hence, the compound showed remark-
able selectivity towards 4-NB, even in the presence of other
potentially interfering aldehydes.
ðI0=IÞ ¼ Ksv ½Aꢀ þ 1
where I0 and I denote the fluorescence intensities of 1′ in the
absence and presence of the analyte, respectively, [A] corres-
ponds to the molar concentration of the analyte, and Ksv rep-
resents the quenching constant.71 In Fig. S33 (ESI†), the S–V
plot for quenching the fluorescence intensity of 1′ by 4-NB is
shown. The value of Ksv for 4-NB, which was determined from
this plot, was found to be 0.59 × 104
M
−1. The S–V plot is
linear in nature in the lower concentration range of 4-NB,
although it shows non-linearity in the higher concentration
range. These results suggest that either a static or dynamic
quenching process occurs in this system.62,71,74,75
Mechanism for the sensing of 4-NB
It is very well known that amine compounds form imines
upon condensation with aldehyde compounds. All the alde-
hydes are supposed to form imine bonds with the –NHNH2
moiety attached to the BDC-NHNH2 ligand. Among all the
selected aldehydes, the electron-withdrawing effect of 4-NB
upon imine formation will be higher as compared to the
others due to the long conjugation of the imine group with the
electron-withdrawing nitro group at the para position. As a
result, the nitro group of 4-NB reduces the electron density
from the imine group much more than the other aldehydes.
Hence, the electron density in the BDC-NHNH2 ligand is
reduced to a larger extent for 4-NB as compared to the other
Fig. 9 Change in the fluorescence intensity of the suspension of 1’
(0.87 mM) (DMSO/HEPES (9 : 1, v/v)) upon the addition of 400 µL of the
50 mM 4-NB solution in the presence of 400 µL of 50 mM solutions of
other potentially competing aldehydes (λex = 360 nm, λem = 430 nm).
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aldehydes. Therefore, the highest fluorescence quenching lity. Either an increase or decrease in fluorescence intensity
3−
effect is observed for 4-NB among all the chosen was observed for PO4 and 4-NB sensing, respectively. The
aldehydes.76,77
mechanism for PO4 sensing involves its coordination with
3−
In order to prove the proposed sensing mechanism, we per- the Zr(IV) ions causing partial collapse of the framework and
formed FT-IR spectroscopy, mass spectrometry and time- subsequent slow release of ligand molecules. For 4-NB
resolved photoluminescence lifetime experiments. In the sensing, the –NHNH2 group of the ligand directly forms a
FT-IR spectrum of untreated 1′ there is no peak at around covalent imine bond with the –CHO group of 4-NB. As a result,
3−
1630 cm−1 whereas there is a new peak at 1637 cm−1 in the IR the response time for PO4 sensing is relatively longer com-
3−
spectrum of 1′ treated with 4-NB (Fig. S4, ESI†). This peak pared to 4-NB detection. The LOD values for PO4 and 4-NB
corresponds to the imine bond formed between the hydrazine sensing were 0.2 and 4.7 µM, respectively.
group of the BDC-N2H3 ligand and the aldehyde group of
4-NB. In the mass spectrum of 1′ (without the treatment of
4-NB), (Fig. S34, ESI†) there is a peak at (+ESI) m/z = 195.0497,
whereas 4-NB treated 1′ (Fig. S35, ESI†) showed a peak at
Conclusions
We have successfully synthesized and characterized a hydrazine-
functionalized Zr(IV) MOF material 1, which has the UiO-66
framework topology. The compound was synthesized by heating
(+ESI) m/z = 328.0631, which corresponds to (E)-2-(2-(4-nitro-
benzylidene)hydrazinyl) terephthalic acid. This observation
confirms that there is an imine product formed by the conden-
a mixture of ZrOCl2·8H2O, H2BDC-N2H3 ligand and benzoic
sation reaction between the hydrazine functionalized BDC
acid (ZrOCl2·8H2O/BDC-N2H3/benzoic acid molar ratio
=
ligand and 4-NB. Hence, from FT-IR spectroscopy and mass
spectrometry, it can be confirmed that the reaction of 1′ with
4-NB forms an imine compound.
1 : 1 : 30) in DMF at 120 °C for 24 h. The activated compound
(1′) showed moderate thermal stability up to 400 °C. It retained
its crystallinity and hence structural robustness when exposed
to water, acetic acid and 1M HCl solutions. As verified by the N2
sorption experiments, the specific BET surface area of 1′ is
818 m2 g−1. We have employed 1′ as a fluorescent turn-on probe
for the selective detection of phosphate (both in vivo and
in vitro) and also as a fluorescent turn-off probe for the selective
sensing of 4-NB in the DMSO/HEPES (9 : 1, v/v) medium. Even
in the presence of interfering intrusive species, the probe
retains its high selectivity. The probe also features high sensi-
tivity. Its detection limits for PO43− ions and 4-NB are 0.196 and
4.7 µM, respectively. Probe 1′ is able to detect phosphate in
living cells. It can also respond towards the difference in the
intracellular phosphate level. The MOF probe can be used
The two quenching mechanisms (static and dynamic) can
be differentiated by studying the time-resolved fluorescence
experiments of the luminescence decay of the fluorophore
compound at different concentrations of the quencher. If the
fluorescence lifetime of the sensor material decreases, the
quenching process is considered as dynamic because
additional relaxation of the excited-state lifetime in this
process results from the collision with the quencher. If the
fluorescence lifetime of the sensor material does not change
after increasing the addition of the quencher, it is regarded as
a static quenching process.75,78,79 Fig. S36 (ESI†) represents the
lifetime decay profile of 1′ before and after the addition of
400 µL of the 50 mM 4-NB solution. As shown in Table S2,
ESI†, the average excited-state lifetimes (〈τ〉*) of 1′ are 2.70 and
1.52 ns before and after the addition of 400 μL of the 50 mM
4-NB solution, respectively. Hence, it can be concluded that
the fluorescence quenching mechanism in this system is
mainly static in nature.
3−
efficiently for determining PO4 ions in real samples such as
tap water, lake water, human urine and human blood serum.
Furthermore, it can be utilized for the in vitro detection of 4-NB
in the fluid secreted from eyes upon exposure to sunlight after
applying the chloramphenicol eye-drop.
Similarities and dissimilarities between PO43− and 4-NB
sensing
Conflicts of interest
3−
The similarity (Table S3, ESI†) between PO4
and 4-NB
There are no conflicts to declare.
3−
sensing involves the high selectivity of 1′ towards PO4 and
4-NB even in the presence of potentially competitive analytes.
The dissimilarities (Table S3, ESI†) observed between the
Acknowledgements
3−
sensing of PO4 and 4-NB involve the sensing medium, the
nature of fluorescence change, response time, sensing mecha-
The authors express their gratitude towards the Science and
Engineering Research Board (SERB; grant no. EEQ/2016/
000012), New Delhi for financial support.
nism and LOD values. We have chosen water or HEPES buffer
3−
(10 mM, pH = 7.4) as the medium for PO4 sensing because
3−
the PO4 ion is highly soluble in these two liquids and it is
highly abundant in environmental water samples. Moreover,
HEPES buffer (pH = 7.4) is known to mimic the physiological
conditions. On the other side, 4-NB sensing was carried out in
a DMSO/HEPES mixture because 4-NB is not soluble in pure
HEPES buffer and hence DMSO was used to increase its solubi-
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