A selective fluorescent 'turn-on' sensor for recognition of Zn2 + in aqueous media

Article abstract of DOI:10.1016/j.saa.2016.02.040

A new rhodamine-based fluorescent probe 'RhAP' was synthesized and successfully characterized using FT-IR, ¹³C NMR and ¹H NMR spectroscopies, LC-MS/MS spectrometry and elemental analysis. The RhAP, a colorless and non-fluorescent compound, showed a selective fluorescent response and colorimetric change for Zn^{2 +} in HEPES buffer (10 mM, EtOH:water, 2:1, v/v, pH 7.2). Upon the addition of two equivalents of Zn^{2 +} to a solution of RhAP, a nearly 35-fold enhancement of the fluorescence intensity, with an emission maximum at 578 nm, was observed in comparison to the sensor alone under the same experimental conditions. The complex formation between RhAP and Zn^{2 +} was found to have a 1:1 ratio based on calculations obtained from Job's plot and the mole ratio plot methods. The results showed that RhAP can be used as an effective fluorescent probe for selective detecting of Zn^{2 +} in an aqueous medium.

Full text of DOI:10.1016/j.saa.2016.02.040

A selective fluorescent ‘turn-on’ sensor for recognition of Zn2 + in aqueous
media
Mecit Ozdemir
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DOI:
Reference:
S1386-1425(16)30097-X
doi: 10.1016/j.saa.2016.02.040
SAA 14306
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14 September 2015
20 February 2016
28 February 2016
Please cite this article as: Mecit Ozdemir, A selective fluorescent ‘turn-on’ sensor for
recognition of Zn2 + in aqueous media, (2016), doi: 10.1016/j.saa.2016.02.040
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ACCEPTED MANUSCRIPT
A selective fluorescent „turn-on‟ sensor for recognition of Zn2+
in aqueous media
Mecit Ozdemira*
Department of Food Processing, Vocational High School, Kilis 7 Aralik University, Kilis, Turkey
* Corresponding author. Tel.: +90 348 814 26 62; fax: +90 348 814 26 63.
E-mail address: mecitozdemir@kilis.edu.tr
ABSTRACT
A new rhodamine-based fluorescent probe „RhAP‟ was synthesized and successfully
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characterised using FT-IR, 13C NMR, H NMR spectroscopies, LC-MS/MS spectrometry and
elemental analysis. The RhAP, a colorless and non-fluorescent compound, showed a selective
fluorescent response and colorimetric change for Zn2+ in HEPES buffered (10 mM, EtOH:water,
2:1, v/v, pH 7.2). Upon the addition of two equivalents of Zn2+ to a solution of RhAP, a nearly
35-fold enhancement of the fluorescence intensity, with an emission maximum at 578 nm, was
observed in comparison to the sensor alone under the same experimental conditions. The
complex formation between RhAP and Zn2+ was found to have a 1:1 ratio based on calculations
obtained from the Job‟s plot and the mole ratio plot methods. The results showed that RhAP can
be used as an effective fluorescent probe for selective detecting of Zn2+ in an aqueous medium.
Keywords: Fluorescent sensor, rhodamine, selective, colorimetric, zinc
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1. Introduction
In recent years, researchers have become increasingly interested in the design and
development of fluorescent sensors based on ion-induced changes in fluorescence due to these
sensors‟ highly selective and sensitive recognition of biologically and environmentally crucial
heavy and transition metal (HTM) ions, and their ability to sense and identify many special metal
species [1]. Zinc is the second most abundant transition metal ion in the human body with
concentrations ranging from nanomolar (nM) to millimolar (mM) [2]. And also, it is an important
cofactor in many biological processes such as neural-signal transmissions and pathology, the
regulation of metalloenzymes, gene transcription, immune function, and mammalian
reproduction. In addition, the disorder of zinc metabolism in biological systems has been
correlated to epilepsy, diabetes, infantile diarrhoea and Alzheimer‟s disease [3]. From this
perspective, the detection of heavy metal ions based on a highly selective and sensitive
fluorescent chemosensor in aquatic ecosystems, drinking water and biological systems is still a
significant research topic due to the serious health issues these ions pose [4]. Although a number
of chemosensors for the detection of zinc have been presently investigated, novel fluorescent
„turn-on‟ probes, which are capable of selectively detecting the presence of zinc ions in biological
and environmental system in aqueous medium under physiological pH conditions, are still a
considerable demand [5]. Therefore, chemists, biologists, clinical biochemists and
environmentalists have recently expressed an interest in the need to design and develop synthetic
receptors that are capable of recognising biologically and environmentally important ion species.
Thus, fluorescent probes that have a turn-on increase or ratiometric response to Zn2+ are
increasingly receiving a great deal of attention, and demand for them is still high [6-9].
As we well known, rhodamine B and its derivatives have commonly been used as an
important class of fluorogenic and chromogenic sensors to visualise metal ions in living cells
without causing cell damage because of their unique properties such as high fluorescence
quantum yields, good photostability, simplicity, low detection limit (LOD), capability for special
recognition and other excellent spectroscopic properties [10]. Rhodamine based chemosensors
are generally non-fluorescent and colorless. However, upon binding with the cation, the ring-
open form of the rhodamine moiety leads to not only strong absorbance and fluorescence
intensity changes, but also it shows an obvious color change during the sensing event, providing
“naked-eye” detection [11-12].
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Herein, we introduce a novel, selective fluorescent turn-on sensor rhodamine-based
fluorescent probe „RhAP‟ based on the metal-coordination induced fluorescent activation (CIFA)
mechanism. The RhAP chemosensor was prepared from a derivative of rhodamine B, which has
a xanthene fluorophore in a spiro-ring configuration. According to the previous reports, the
spirolactam moiety of the sensor prepared from a rhodamine derivative is expected to act as a
signal switcher, which is visualized to turn on upon binding with metal cations [5, 13-15].
Consequently, the proposed RhAP chemosensor displayed a selective fluorescent response and
visible change from colorless to purple. Fluorescence spectroscopic study of the sensor‟s
interaction with Zn2+ also showed that RhAP is highly selective toward zinc more so than other
competing metal ions under identical conditions.
2. Experimental Procedure
2.1. Equipment and reagents
Unless otherwise specified, chemicals were purchased from commercial suppliers and
used without further purification. 2-Acetylpyridine and metal salts (nitrate or chloride) were
purchased from Sigma-Aldrich and Merck. All the solvents were of analytic grade and water was
redistilled. Rhodamine hydrazine was synthesized according to the method outlined in a previous
report [16]. Structural characterization of the RhAP was carried out using Fourier transform
infrared (FT-IR), hydrogen-1 nuclear magnetic resonance (1H NMR), carbon-13 nuclear
magnetic resonance (13C NMR), liquid chromatography–mass spectrometry (LC-MS/MS),
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elemental analyses, fluorescence spectra and UV-Vis spectra techniques. The H NMR and C
NMR spectra were recorded on a Bruker AVANCE III HD 400 NMR spectrometer using
deuterated chloroform (CDCl3) as the solvent and tetramethylsilane (TMS) as an internal
standard. LC-MS/MS analyses were conducted using a Varian 325-MS detection system. FT-IR
spectra were measured using a PerkinElmer Spectrum 100 spectrometer. Absorption spectra were
measured using a Varian Cary 100 Bio spectrophotometer at 298 K. All fluorescent
measurements were conducted using a Varian Cary eclipse fluorescence spectrophotometer at
298 K. The pH measurements were determined using a Corning pH meter equipped with a
Sigma-Aldrich micro combination electrode calibrated with a standard buffer solution.
To prepare the stock solutions of metal ions, the nitrate or chloride salts, Na+, K+, Ca2+,
Mg2+, Cr3+, Fe3+, Hg2+, Cu2+, Pb2+, Zn2+, Fe2+, Co2+, Ni2+, Cd2+, Al3+, Mn2+ and Ag+, were
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dissolved in double-distilled water to create 1 mM aqueous solutions. A 1 mM stock solution of
RhAP was prepared in EtOH. These solutions were further diluted to the micro-molar (µM) level
for fluorescence and absorption spectroscopy measurements with an EtOH-water (2:1, v:v) buffer
(10 mM, HEPES, pH 7.2). Although the stock solution of RhAP used in the experimental studies
was stored for more than one month under daylight conditions, no changes in spectroscopic
properties were observed.
2.2. Synthesis of RhAP
The RhAP was synthesized using the procedures previously reported in [17]. In short, 2-
acetyl-pyridine (242 mg, 2 mmol) in ethanol, with a few drops of acetic acid, was slowly added
to the ethanolic solution of rhodamine hydrazide (912 mg, 2 mmol). The mixture was stirred and
heated to reflux overnight. The crude product evaporated under the reduced pressure and the
mixture was purified by silica gel column chromatography using CH2Cl2/CH3OH (20:1, v/v) as
the eluent to afford a 0.86 g pure product, a 76 % yield of colorless RhAP.
Elemental Anal. (Calcd./Found): % C (74.97/74.68), % H (6.28/6.32), % N (12.87/12.80).
FT-IR (RhAP, ν/cm-1): 3084(vw, νAr-H), 2967-2923(w, νC-H), 1691(vs, νC=O); 1614(vs, νC=N
,
azomethine), 1548(m, Py, νC=N), 1514(vs, Ar, νC=C), 1426, 1304, 1216, 1116(C-O); 1079, 815,
785, 755, 698, 674, 569, 531(Supplemental Fig. S1). FT-IR (RhAP+Zn2+, ν/cm-1): 3086 (vw, νAr-
H), 2973-2930(w, νC-H), 1645(w, νC=O), 1586(vs, νC=N, azomethine), 1508(w, Py, νC=N), 1466(m,
Ar, νC=C), 1466, 1411, 1335, 1272, 1246, 1180, 1011, 922, 821, 785, 683, 496 (Supplemental Fig.
1
S2). H NMR (CDCl3-d, 400 MHz) δ(pmm): 8.52(d, 1H), 7.8(d, 1H), 7.5(t, 2H), 7.48(m, 2H),
7.19(d, 2H), 6.59(d, 2H), 6.3(s, 2H), 6.28(d, 2H), 3.33(q, 8H), 2.37(s, 3H), 1.15(t, 12H)(
Supplemental Fig. S3). 13C NMR (CDCl3-d, 400 MHz) δ(pmm): 169(C17), 161(C24), 160, 159,
155, 153,151, 148, 144, 136, 132, 131, 128, 124, 123, 122, 108, 106, 97, 77(solvent),
44,5(C34,35,39,40), 17,51(C25), 12,86(C35,37,41,42) (Supplemental Fig. S4). ESI-MS: The
mass spectrum of the chemosensor was represented in Supplemental Fig. S5. Fragments at m/z =
582 (base peak) was attributed to molecular ion peak [C35H37N5O2 + Na] +. The other one at m/z
= 560 appeared in the mass spectra was attributed to [C35H37N5O2 + H] +. In addition to those
peaks, there are also fragmentation products of molecular ion such as 423, 236, 183, and 101.
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Scheme 1. Synthesis of the chemical probe RhAP
2.3. Determination of the binding mode (S:M), dissociation constant (Kd) and limit of detection
(LOD)
The stoichiometry of RhAP with Zn2+ was determined using the Job‟s plot and the mole ratio
plot methods. The absorption enhancement observed at 562 nm was plotted against the molar
value of the [Zn2+]/ [Zn2+] + RhAP and [Zn2+]/RhAP. The maximum absorption enhancements
were obtained at 0.23 (Job‟s plot calculation) and 3.1 (mole ratio plot calculation). The
dissociation constant of the RhAP sensor was determined using the Benesi-Hildebrand equation
(Eq. 1) by employing fluorescence titration data. For the practical application, the LOD of the
RhAP to Zn2+ was also calculated by using IUPAC equation [18].
3. Results and Discussion
The RhAP chemosensor was designed and prepared from rhodamine hydrazide and 2-
acetylpyridine in ethanol, with a good yield. The compound was successfully characterised using
elemental analysis, FT-IR, 1H-NMR, 13C-NMR and LC-MS/MS methods. All the
characterization studies confirmed the structure of the RhAP, which is a colorless powder that is
highly stable in ethanol. Moreover, the geometry of RhAP was optimised using the density
functional theory (DFT) method with the 6-31G (d, p) basis set in the ground state. Molecular
electrostatic potential (MEP) and frontier molecular orbitals (FMOs) of the fluorescent probe
prepared in this study were investigated using theoretical calculations (Supplemental Figs. S6 and
S7) [9, 19].
In order to better understand the fluorescent sensor‟s ability to distinguish between the sensor
and the zinc ions in HEPES buffered (10 mM, EtOH:water, 2:1, v/v, pH 7.2), fluorescence and
UV-vis spectroscopy techniques that were previously reported in the literature were used. The
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stock solutions of the sensor and the metal ions were prepared at a concentration of 1 mM, and it
was diluted to the micro-molar (µM) level [20-21].
3.1. Binding mode and sensing mechanism
According to the previously reported mechanism for some types of RhAP chemsensors, it is
possible that the metal cations coordinate with the corresponding atom of the rhodamine group
and cause the spirolactam ring to open [22-26]. This indicates that the carbonyl oxygen atom of
spirolactam moiety plays an important role in binding RhAP to Zn2+ [27-30]. In order to shed
some light on the binding mode for the complexation between RhAP and Zn2+, IR and 1H NMR
spectra experiments were carried out, which indicated the coordination of the atoms (ONN) of
receptor to zinc by opening of the spirolactam ring. IR spectra of RhAP and RhAP-Zn2+ were
measured in ATR method, respectively, and the results were shown in Fig. 1. The characteristic
stretching peaks at 1691.43 cm-1, which corresponds to the carbonyl (νC=O) absorption of RhAP,
was shifted to a lower frequency (1645 cm-1) upon chelating with Zn2+, showing that a strong
polarization of the C=O bond and also the cleavage of N-C bond in the spirolactam ring by the
effective interaction of the RhAP with Zn2+. Also, the stretching bands corresponding to the νC=N
(azomethine) and νC=N (pyridine) in the RhAP were recorded at 1614.31 and 1548 cm-1,
respectively. Those of characteristic absorption bands of free RhAP, in cm-1, were also shifted to
lower wave numbers by 25 to 40 cm-1 on complexation. The IR spectrum of the RhAP + Zn2+
apparently gives evidence of coordination of the azomethine „N’ and the pyridine „N’ atoms and
of the amidic carbonyl „O‟ atom [31-33].
Fig. 1
1
To further support the binding structure, H NMR spectra of RhAP was recorded in the
absence and presence of the 0.5 and 1.1 equiv. amount of the zinc salt dissolved in water, as
1
depicted in the supplemental Fig. S8. In the H NMR spectra, the signals of RhAP for the
different types of assigned protons that are in the aromatic and aliphatic regions in the presence
of zinc ions showed chemical shift changes. The RhAP-Zn2+ complex signal pattern of the
aromatic and aliphatic protons also demonstrates the involvement of the pyridine moiety of the
probe unit of RhAP in the binding of Zn2+. According to these findings, a possible binding mode
for RhAP with Zn2+ was proposed as presented in Scheme 2 [34-36].
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Based on these determinations, the change in color can also be related to the structural
transformation of RhAP from the spirolactam (non-fluorescent) form to the ring-opened
(fluorescent) form owing to its complexation with Zn2+. Thereby, as shown in Scheme 2 and Fig.
2, the colorless solution turned purple, which provided “naked-eye” detection of Zn2+ due to the
ring opening of spirolactam of the sensor, when Zn2+ was added to the RhAP in the HEPES
buffer solution. However, the addition of other competing metal ions did not lead to changes in
the color of the RhAP solution in the absence of Zn2+, except when the competing metal ions,
Co2+, Ni2+ and Cu2+ were present.
Scheme 2. The proposed binding mechanism of RhAP induced by Zn2+ in HEPES buffered (10 mM, EtOH:water,
2:1, v/v, pH 7.2). The insert shows the change of the color with the addition of Zn2+ to RhAP in buffered solution.
Figs. 2
3.2. Selectivity of the Sensor
To further investigate the cation binding properties of RhAP, its sensitivity was determined
and selectivity studies for Zn2+ ions were conducted using UV-vis and fluorescence spectroscopy
techniques with various possible interfering metal ions, as shown in Figs. 3 and 4. The absorption
spectra of free RhAP in an EtOH-water solution displayed almost no absorption between 450 nm
and 600 nm. Except for absorption increases for Zn2+, Co2+, Ni2+ and Cu2+, other competing metal
ions did not cause considerable absorption changes of RhAP, as presented in Fig. 3. This was
attributed to the closed (spirolactam) forms of the sensor molecules in the solution. As depicted
in Fig. 2, only Zn2+, Co2+, Ni2+ and Cu2+ induced a notable color change in the buffer solution
under visible light, which can be ascribed to the spirolactam bond cleavage of the fluorophore
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group, while the other metal ions did not show any obvious response [37-42]. Furthermore, the
addition of Na+, K+, Ca2+, Mg2+, Cr3+, Fe3+, Al3+, Mn2+, Hg2+, Cu2+, Pb2+, Fe2+, Co2+, Ni2+, Cd+2
and Ag+ exerted little or no effect on the emission intensity of RhAP, while remarkable
fluorescence enhancement was detected with the addition of Zn2+ by excitation of the fluorescent
sensor at 535 nm. As depicted in Fig. 4, RhAP showed high fluorescence selectivity towards
Zn2+ over the other metal ions, with the exception of a small amount of fluorescence enhancement
for Fe2+ and Pb2+ (II). Meanwhile, when Co2+, Ni2+ and Cu2+ ions were added to the sensor
solution, significant changes in UV-vis spectra were observed. However, the fluorescence
intensity did not increase when those metal ions were added because of their well-known
paramagnetic properties. They have unfilled d shells, which might quench the fluorescence of
sensor via electron or energy transfer [43-45].
To further explore the ability of probe RhAP to sense Zn2+, fluorescence titration experiments
on the sensor solution with Zn2+ were conducted in an EtOH-water solution under physiological
pH conditions. As shown in Supplemental Fig. S9, the fluorescence intensity steadily increased
when Zn2+ was progressively added to the RhAP solution. From the titration profiles, the
dissociation constant (Kd) and the LOD of RhAP for Zn2+ were determined.
Thanks to the fluorescence response of RhAP toward Zn2+ in the presence and absence of
ions, such as Na+, K+, Ca2+, Mg2+, Ag1+, Cr3+, Fe3+, Al3+, Mn2+, Hg2+, Cu2+, Pb2+, Fe2+, Co2+, Ni2+
and Cd+2, comparative experiments of zinc with various other metal ions were also conducted as
given in Supplemental Fig. S10. The reduced selectivity for Zn2+ did not change with the addition
of the other metal ions; only Fe (II) and Pb (II) showed a very small increase in fluorescence
intensity. Furthermore, Supplemental Fig. S10 also reveals that some alkaline, alkaline-earth or
transition metal ions do not interfere with the Zn2+-induced fluorescence increase. This
demonstrates that the proposed RhAP chemosensor has a good selectivity and sensitivity toward
Zn2+.
In addition, the reversibility of the fluorescent probe has been a significant aspect in practical
applications. The coordination that occurs between RhAP and Zn2+ was found to be reversible
using ethylenediamminetetraacetic acid (EDTA) titration, as supported in Fig. 5 and Scheme 2.
Once the RhAP-Zn2+ complex was finally treated with 0.3 mM EDTA, the purple solution turned
colorless, which confirmed that the coordination of this fluorescent probe with Zn2+ is reversible
by chemical processes [33, 46].
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Figs. 3, 4, and 5
3.3. The effect of pH on fluorescence intensity
The optimum pH conditions for the successful application of the RhAP and RhAP-Zn2+
complex were carefully investigated. For the purpose of this assay, as shown in Fig. 6, the effects
of pH on the fluorescence intensity of the RhAP chemosensor were measured in the absence and
presence of Zn2+ in HEPES buffered (10 mM, EtOH:water, 2:1, v/v, pH 7.2). The experiments
were conducted at a pH ranging from 4.0 to 11.0. No change in fluorescence intensity was
observed in the free chemosensor in an EtOH-water solution when the pH ranged between 7.0
and 11.0. However, small changes in fluorescence intensity were observed under acidic
conditions (pH<7). In contrast, the result that occurs when two equivalents of Zn2+ are added to a
solution of RhAP, a red-shifted emission peak at 578 nm was observed in this experiment, with
an important enhancement of fluorescence over a comparatively wide pH range (6.0–11). This is
due to the spirolactam ring opening as a result of the complexation reaction between the sensor
and Zn2+. However, none of the other competing metal ions led to significant changes in
fluorescence intensity under identical conditions. In consequence, the results suggest that RhAP
can be further used as a selective fluorescent turn-on sensor to detect zinc species under
physiological pH conditions [16, 18].
Figs. 6
3.4. Determinations of stoichiometry, dissociation constant (Kd) and limit of detection (LOD)
In the proposed system, the stoichiometry of RhAP-Zn2+ was obtained by using the Job‟s plot
and mole ratio plots methods, as depicted in Fig. 7 and Supplemental Fig. S11 [47]. To determine
the dissociation constant of RhAP, different amounts of Zn2+ ions were titrated into the ethanol-
water solution that contained the fluorescent probe. As shown in Supplemental Fig. S9, the
addition of various concentrations of Zn2+ (ranging from 0–180 μM) changed the emission
intensity of RhAP. In this titration experiment, a gradual enhancement in the emission intensity
of RhAP was observed when Zn2+ ions were added to the solution [48]. The dissociation constant
(Kd) value of Zn2+ with RhAP was estimated from the emission intensity data following the
modified Benesi–Hildebrand equation. [9]. According to the Job‟s plot assay, assuming a 1:1
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binding mode between RhAP and Zn2+, the dissociation constant of the RhAP chemosensor with
zinc ions was determined from the plot of the linear regression of log[(F-Fmin)/(Fmax-F)] versus
log[M] in equation to find the intercept as „logK‟ and the slope as „n‟ by using Eq. 1, as follows:
log[(F-Fmin)/(Fmax-F)] = logK + nlog[M]
Where F is the observed fluorescence and Fmax is the maximum fluorescence for the RhAP-Zn2+
complex at an excess amount of zinc ions. Fmin is the fluorescence of the sensor in absence of
Zn2+ and „n‟ is the number of Zn2+ bound per RhAP. [M] is the concentration of the zinc ions
(µM) added to the RhAP solution. Based on the Benesi-Hildebrand equation, the dissociation
constant (Kd) obtained from the intercept of the linear plot was found to be Kd = 3.16x10-7 M
(Fig. 8) [49]. To better demonstrate the selectivity of RhAP towards the Zn2+ ions, a protocol to
determine the LOD was performed using fluorescence titration data with the equation, based on
the IUPAC definition (LOD = 3Sb/m), where Sb is the standard deviation of the blank
measurements and m is the slope between the fluorescence intensity and the zinc concentrations.
Thus, the LOD of RhAP with Zn2+ was found to be 29 nM, as shown in Fig. 9 [19, 50-51].
Figs. 7, 8 and 9
4. Conclusions
In summary, this paper reports on a rapid and selective fluorescence turn-on detection system
for Zn2+ based on a rhodamine hydrazone derivative. Overall, the results presented in this study
demonstrated that RhAP shows highly selective fluorescent enhancement with apparent color
change from colorless to purple which provides „naked-eye‟ detection of Zn2+ ions in an EtOH-
water solution. It also found that the fluorescence intensity of RhAP at 578 nm displayed a
quantitative response to Zn2+. In addition, RhAP exhibited a very low detection limit of 29 nM
zinc as compared to sensors that have similar coordination moiety. Based on these findings,
RhAP is a promising fluorescent probe for detecting Zn2+ metal ions in aqueous media under
physiological pH conditions. Consequently, the proposed novel RhAP might also have a
practical application for the selective detection of Zn2+ in drinking water and human serums.
Acknowledgements
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In this paper, Gaussian 09W used for theoretical calculations was supported by Kilis 7 Aralik
University with BAP project 2010/02/08.
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Figures
Fig.1. IR spectra shows that RhAP before (a) and after (b) addition of Zn2+
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Fig.2. Color changes of RhAP (50 µM) in absence and presence of Na1+, K1+, Mg2+, Ca2+, Al3+, Ag1+, Zn2+, Hg2+,
Fe2+, Cd2+, Mn2+, Ni2+, Cu2+, Fe3+, Cr3+, Pb2+, Co2+ (100 µM) in HEPES buffered (10 mM, EtOH:water, 2:1, v/v, pH
7.2) under visible light (a) and UV lamp at 365 nm (b).
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Fig.3. UV-vis spectra of RhAP (50 µM) in HEPES buffered (10 mM, EtOH:water, 2:1, v/v, pH 7.2) with 2 equiv. of
metal ions: Na1+, K1+, Mg2+, Ca2+, Al3+, Ag1+, Zn2+, Hg2+, Fe2+, Cd2+, Mn2+, Ni2+, Cu2+, Fe3+, Cr3+, Pb2+ and Co2+
The bar graph shows the changes of relative absorbance value upon treatment with same metal ions at 562 nm.
.
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Fig.4. Fluorescence emission spectra of RhAP (50 µM) in HEPES buffered (10 mM, EtOH:water, 2:1, v/v, pH 7.2)
with 2 equiv. of metal ions: Na1+, K1+, Mg2+, Ca2+, Al3+, Ag1+, Zn2+, Hg2+, Fe2+, Cd2+, Mn2+, Ni2+, Cu2+, Fe3+, Cr3+,
Pb2+ and Co2+; Insert: Photo picture under visible light and portable UV lamp at 365 nm (a), bar graph representing
the changes of relative emission intensity of RhAP at 578 nm upon treatment with same metal ions (λ ex = 535 nm)
(b).
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4.0
3.5
3.5
3.0
3.0
2.5
2.5
2.0
1.5
2.0
1.0
0.5
1.5
0.0
1.0
0
50
100
150
200
250
300
[EDTA] (M)
0.5
0.0
450
500
550
600
650
700
750
Wavelength (nm)
Fig.5. UV-vis spectra changes of RhAP+Zn2+ with increasing concentration of EDTA (2 equiv., 0 to 300 µM, from
top to bottom) in HEPES buffered (10 mM, EtOH:water, 2:1, v/v, pH 7.2). The inset exhibits the absorbance at 562
nm as a function of EDTA concentration.
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500
2+
RhAP + Zn
RhAP
400
300
200
100
0
4
5
6
7
8
9
10
11
12
pH
Fig.6. The effect of pH (4.0-11.5) on the relative fluorescence intensity of RhAP (50 µM) in the absence ( ) and
presence ( ) of Zn2+ (1 equiv.) in HEPES buffered (10 mM, EtOH:water, 2:1, v/v, pH 7.2). The pH values of solution
were adjusted by using an appropriate volume of 0.1 M HCl or NaOH stock solution.
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0.25
0.20
0.15
0.10
0.05
0.00
0.20
0.18
0.16
0.14
0.12
0.10
0.08
0.06
0.04
0.02
0.00
0.0
0.2
0.4
0.6
0.8
1.0
2+
2+
[Zn ] / [Zn ] + [RhAP]
450
500
550
600
650
700
Wavelength (nm)
Fig.7. Job‟s plot established from the absorption spectra of RhAP and Zn2+ under a constant total concentration. The
inset shows the absorbance at 562 nm as a function of {[Zn2+]/[Zn2+]+[RhAP]} value.
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1.4
2+
RhAP - Zn
y = 0.92358*x - 0.56324
1.2
2
R
= 0.99454
1.0
0.8
0.6
0.4
1.0
1.2
1.4
1.6
1.8
2.0
2+
lg{[Zn ]/(M)}
Fig.8. Benesi-Hildebrand plot (fluorescence intensity at 578 nm, λex = 535 nm) of RhAP, assuming a 1:1 binding
mode for association between RhAP and Zn2+.
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2+
RhAP - Zn
1000
y = 20.43133*x - 2.47057
2
R = 0.99492
800
600
400
200
0
0
10
20
30
40
50
2+
[Zn ] ()
Fig.9. Fluorescence intensity at 578 nm of RhAP upon continuous addition of increasing amount of Zn2+ (0-50 µM)
in HEPES buffered (10 mM, EtOH:water, 2:1, v/v, pH 7.2, λex = 535 nm).
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Graphical Abstract
Proposed binding mode of sensor RhAP and Zn2+ in EtOH-water (2:1, v/v) solution
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Highlights
 Synthesis and characterization of a new fluorescent sensor „RhAP‟
 The selectivity of RhAP for Zn2+ was investigated by spectroscopic techniques
 The results showed that the sensor may be further performed for the selective detection of
Zn2+ in aqueous media
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