- Email: [email protected]
Cu2+ and its application in bioimaging
Accepted Manuscript Title: A novel solvent-dependently bifunctional NIR absorptive and fluorescent ratiometric probe for detecting Fe3+ /Cu2+ and its application in bioimaging Author: Shao Li Di Zhang Xinyu Xie Saige Ma Yao Liu Zhanhui Xu Yanfeng Gao Yong Ye PII: DOI: Reference:
S0925-4005(15)30552-9 http://dx.doi.org/doi:10.1016/j.snb.2015.10.086 SNB 19230
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
14-8-2015 17-10-2015 24-10-2015
Please cite this article as: S. Li, D. Zhang, X. Xie, S. Ma, Y. Liu, Z. Xu, Y. Gao, Y. Ye, A novel solvent-dependently bifunctional NIR absorptive and fluorescent ratiometric probe for detecting Fe3+ /Cu2+ and its application in bioimaging, Sensors and Actuators B: Chemical (2015), http://dx.doi.org/10.1016/j.snb.2015.10.086 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A novel solvent-dependently bifunctional NIR absorptive and fluorescent ratiometric probe for detecting Fe3+/Cu2+ and its application in bioimaging
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Shao Lia, Di Zhangb*, Xinyu Xiec, Saige Maa, Yao Liua, Zhanhui Xua, Yanfeng
Phosphorus Chemical Engineering Research Center of Henan Province, the College
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a
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Gaoc, Yong Yea*
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of Chemistry and Molecular Engineering, Zhengzhou University, Zhengzhou 450052, China.
Institute of Agricultural Quality Standards and Testing Technology, Henan Academy
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b
of Agricultural Sciences, Zhengzhou 450002, China.
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The School of Life Sciences, Zhengzhou University, Zhengzhou 450001, China.
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c
∗
Corresponding author; Email: [email protected] (Di Zhang); [email protected] (Yong Ye) 1
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Abstract: A novel rhodamine-cyanine based probe LS1 was designed and synthesized, which can act as a bifunctional NIR ratiometric colorimetric and fluorescent probe for detecting Fe3+ and Cu2+ in solvent of MeOH/H2O and MeCN/H2O respectively. As expected, it exhibited high selectivity and sensitivity for detecting Fe3+ and Cu2+ over
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other commonly coexistent metal ions in their respective systems with a broad pH span. The detection limit was measured to be 0.737 µM for Fe3+ and 1.019 µM for
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Cu2+. Furthermore, fluorescence imaging experiments of Cu2+ ions in living SH-SY5Y109 cells demonstrated its value of practical application in biological
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systems.
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Keywords: NIR, fluorescent probe, Fe3+, Cu2+, Cyanine, Rhodamine 1. Introduction Recently, the design and development of chemosensors for sensing and recognition of environmentally and biologically important heavy- and transition-metal ions has attracted wide-spread interests of biologists, chemists, clinical biochemists [1-5]
. Among transition metal ions, iron is the most abundant
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and environmentalists
essential trace element for both plants and animals. Fe3+ plays an important role in
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enzyme catalysis, cellular metabolism, and as an oxygen carrier in hemoglobin and a
cofactor in many enzymatic reactions involved in the mitochondrial respiratory chain [6] [7]
, Parkinson’s disease
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. Both of its deficiency and excess can result in serious disorders such as Huntington [8]
. And copper, rank only to iron and zinc, is the third most
abundant essential trace element in the human body, and performs some important
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roles in many fundamental physiological processes in organisms [9], but excessive levels of cooper have malign influences [10-12]. Therefore, many excellent works of reported and investigated [13-19].
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Fe3+ and Cu2+ sensing by synthesized colorimetric/fluorescent probes have been Most of these chemosensors have the absorption and emission in the
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ultraviolet-visible (UV/Vis) light rage, which renders them difficult to be employed
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for sensing and imaging targets of interest in living lives, as the absorption and auto-fluorescence of biomolecules in UV/Vis region are high [20]. Recently, cyanine dyes, as a long wavelength (NIR region at around 650-900 nm) analyte-responsive fluorescent probes, have become the focus of analytical and biological. As it has minimum photo-damage to biological samples, deep tissue penetration, and minimum interference form background auto-fluorescence by biomolecules in the living systems, cyanine dyes have been well used in vivo fluorescence imaging [21-26]. And as the heptamethine cyanine has a rigid chlorocyclohexenyl ring in the polymethine chain that could increase its photostability, enhance the fluorescence quantum yield, and provide an ideal site for further modification with amino or phenol substitutions, we choose heptamethine cyanine as a fluorophore along with other cyanine analogues. According to our knowledge, only few NIR analyte-responsive probes based on heptamethine cyanine for sensing metal ions have been reported [27-36]. However, in the detecting of most probes, the absorbance and fluorescence intensity measurements may be influenced by the variations in the assay sample 3
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environment (pH, concentration, temperature, and so forth), due to the only optical signal change. To eliminate those effects, a ratiometric colorimetric/fluorescent [37-39]
measurement is desirable
. Because ratiometric chemosensors can provide a
built-in correction for environmental effects by simultaneous measurement of two fluorescence signals at different wavelengths followed by calculation of their intensity ratio
[40-41]
. Although some ratiometric chemosensors have been constructed recently,
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there barely are reports on the ratiometric sensing of metal ions in the near-infrared (NIR) region. Thus, there is still an intense demand for new ratiometric optical probes
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for Fe3+/Cu2+ with favorable photophysical proprieties in the NIR region.
Tuning metal ion selectivity by altering the solvent system is in keep with the
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newly emerged chemosensor design concept of “single sensor for multiple analytes”, namely, analysis of more than one analyte by one receptor using a single or an array of detection method
[42-45]
. In this paper, we reported a novel probe LS1, based on
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cyanine-coupled rhodamine B, that could detect Fe3+ and Cu2+ ions in two kinds of
2. Experimental 2.1 Apparatus
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solvent systems separately with high selectivity and sensitivity.
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Absorption spectra were measured on a on a UV-2102 double-beam UV/VIS
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spectrometer, Perkin Elmer precisely. Fluorescence spectra were recorded on the F-4500 FL Spectrophotometer, and the excitation and emission wavelength band passes were both set at 10.0 nm. The pH was measured with a Model pHs-3C meter (Shanghai, China). 1H and 13C NMR spectra were recorded using a Bruker DTX-400 spectrometer. Samples were dissolved in CDCl3 and placed in 5 mm NMR tubes. TMS was used as internal reference. ESI mass spectra were carried out on an HPLC Q-Exactive HR-MS spectrometer (Thermo, USA) by using methanol as mobile phase. Fluorescence images experiments were carried out with a Nikon-80i inverted fluorescence microscope. 2.2 Materials All chemical reagents were used as received from commercial sources without further purification. Solvents for chemical synthesis and analysis were purified according to standard procedures. Deionized water was used throughout the experiment. Chloride salts of metal ions (Li+, K+, Na+, Ca2+, Mg2+, Ba2+, Zn2+, Fe2+, Mn2+, Cu2+, Co2+, Ni2+, Cd2+, Cr3+, Hg2+, Al3+) and the nitrate salts of Ag+, Pb2+ and 4
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Fe3+ ions were prepared as 10.00 mM in water solution. 2.3 Synthesis N
O
N
N O
N
N Cl
+
H N
N EtOH O
ref lux, 8 h
N NH2
NI
O
N I
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N
LS1
2
1
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Scheme 1. Synthetic route of probe LS1.
Compound 1 and 2 was synthesized by reported methods [46].
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Compound 2 (0.61 g, 1 mM) was dissolved in anhydrous ethanol (20 mL) in a round bottom flask, then compound 1 (0.97 g, 2 mM) was added. The mixture was
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heated to reflux, and stirred under N2 atmosphere for 8 h. Then the solvent of resulting mixture was evaporated in vacuum. The residue was purified by silica gel thin layer chromatography with EtOAc/MeOH (8:1, v/v) to obtain 0.46 g of dark blue 1
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solid in a yield of 43 %.
H NMR (400 MHz, CDCl3, ppm) δ: 1.14 (t, 12 H, J = 7.0 Hz), 1.30 (t, 2 H, J = 13.6
Hz), 1.52 (s, 12 H), 1.67 (t, 2 H, J = 6.0 Hz), 2.41 (bs, 4 H), 2.90 (t, 2 H, J = 14.0 Hz),
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3.08 (s, 1 H), 3.31 (m, 8 H), 3.49 (s, 3 H), 5.19 (d, 2 H, J = 12.4 Hz), 6.18 (d, 1 H, J =
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2.4 Hz), 6.20 (d, 1 H, J = 2.4 Hz), 6.35 (d, 2 H, J = 2.3 Hz), 6.43 (d, 2 H, J = 8.8), 6.57 (d, 2 H, J = 7.8 Hz), 6.80 (t, 2 H, J = 7.4 Hz), 7.10 (m, 6 H), 7.40 (t, 2 H, J = 3.5 Hz), 7.91 (t, 1 H, J = 4.6 Hz);
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C NMR (100 MHz, CDCl3, ppm) δ: 12.65, 14.12,
21.41, 22.69, 26.33, 28.62, 29.69, 44.34, 44.41, 93.62, 97.87, 97.98, 104.77, 107.49, 108.00, 108.24, 121.63, 121.73, 122.73, 124.08, 127.51, 128.02, 128.28, 128.47, 128.82, 132.92, 139.60, 144.11, 148.88, 148.92, 153.41, 153.77; HR-MS m/z: Calcd for C62H71N6O2+ [M-I-]+ 931.5633, found 931.5667. 3. Results and analysis
Probe LS1 was dissolved in MeCN or MeOH to make a 1 mM stock solution, respectively. Then the stock solution was further diluted to require concentration for measurement. 3.1 The detection of Fe3+ in H2O-MeOH solution. The UV-vis and Fluorescence characteristics of probe LS1 in a solution of H2O-MeOH (4:1, v/v) have been studied. As shown in Fig. 1 and Fig. S4, probe LS1 5
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(10 μM) exhibited obviously selective response to Fe3+ among 19 tested metal ions (Li+, K+, Na+, Ca2+, Mg2+, Ba2+, Zn2+, Fe2+, Mn2+, Pb2+, Cu2+, Ag+, Fe3+, Co2+, Ni2+, Cd2+, Cr3+, Hg2+, Al3+). When there were no metal ions being added, the solution of probe LS1 displayed a single strong absorption band at about 650 nm, which is attributed to the absorption of cyanine moiety. After all the 19 metal ions (100 μM) were added separately into the solution of probe LS1 and placed for 30 min, only the
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solution containing Fe3+ changed its color from blue to red obviously which allowing
naked-eye detection. In the meantime, a large range of hypochromatic shift (136 nm)
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increased at 514 nm, allowing colorimetric detection of Fe3+.
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in absorption spectrum could be observed as the peak decreased at 650 nm and
Fig. 1. Absorbance spectra of LS1 (10 μM) in the absence and presence of 10 eq. different metal ions in MeOH/water (1/4, v/v) solution. Inset shows the photo of LS1with different metal ions.
The free probe LS1 exhibited an emission band at about 777 nm attributed to
cyanine moiety and another emission band at about 577 nm attributed to rhodamine moiety (Fig. S4). A prominent enhancement of characteristic fluorescence of rhodamine B emerged at 577 nm after 10 eq. of Fe3+ was added into the solution. Meanwhile, under the same condition, no obvious response could be observed upon the addition of the same amount of other ions. A mild increase of fluorescence at 577 nm was also detected when the same amount of Al3+ was added due to their low binding affinity to probe LS1. It was noteworthy that the difference between the two emission wavelength was quite large (almost 200 nm), which not only contribute to the accurate measurement of the intensities of these two emission peaks, but also resulted in a large ratiometric value. As shown in Fig. 2, the solution of free probe LS1 exhibited a very low fluorescence intensity ratiometric value, but upon the 6
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addition of 10 eq. of Fe3+, there was prominent enhancement of this value. In the meantime, under the same condition, the addition of the other ions had almost no response to this value, except a mild fluorescence enhancement factor was also detected for Al3+. Furthermore, as shown in Fig. 3, the competitive experiment also confirmed that the background metal ions showed very low interference with the detection of Fe3+ in
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the solution of H2O-MeOH (4:1, v/v). It was also investigated that the ratiometric fluorescence signal response of probe LS1 toward Fe3+ in the presence of various
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coexistent anions such as NO3-, I-, Cl-, Br-, H2PO4-, SO42-, HCO3- and AcO-, which revealed that all the tested anions have little interference on the detecting of Fe3+
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(Fig.S5). Therefore, these results suggested that probe LS1 has a high selectivity for
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Fe3+ in the presence of these tested foreign metal ions and anions.
Fig. 2. Fluorescence intensity ratio (F577/F777) of LS1 (10 μM) in the presence of 10 equiv different metal ions in MeOH/water (1/4, v/v) solution. λex = 520 nm, scan range 540–800 nm, slit width 10 nm.
Fig. 3. Fluorescence intensity ratio (F577/F777) of LS1 (10 μM) upon addition of 10 equiv Fe3+in the presence of 10 equiv background metal ions in MeOH/water (1/4, v/v) solution. 7
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To further investigate the interaction between Fe3+ and probe LS1, an ultraviolet tritration experiment was carried out (Fig. 4). Upon addition of increasing concentration of Fe3+ ions to the solution, a new absorption band centered at 514 nm appeared while the absorption band centered at 650 nm disappeared, which could be ascribed to the Fe3+ induced spirocyclic-pening reaction accompanied with activating
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the photo-induced electron transfer (PET) process of cyanine to Fe3+ metal ion. A Job’s plot (Fig. 4 inset) analysis corresponding to 0.50 the concentration ratio
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indicated the 1:1 stoichiometric ratio between probe LS1 and Fe3+.[47]
Fig. 4. Absorption spectra of LS1 (10 µM) with gradual addition of various amounts of Fe3+ (from bottom 0-6 eq) in MeOH/water (1/4, v/v) solution. Inset shows Job's plot for determining the stoichiometry of LS1 and Fe3+, ([LS1] + [Fe3+] = 100 μM).
To investigate the practical applicability of probe LS1, the detection limit of this
new chemosensor was evaluated. The fluorescence titration profile of probe LS1 (10 μM) with various concentration of Fe3+ was recorded at excitation wavelength of 520 nm (Fig. 5). The linear response of the emission intensity ratio (F577/F777) toward Fe3+ was obtained in Fe3+ concentration of 0-20 μM, and the detection limit (3σ/slope) of probe LS1 for the determination of Fe3+ was found to be 0.737 µM (Fig.5. inset) [48-49]
.
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Fig. 5. Fluorescence emission spectra of compound LS1 (10 μM) in the presence of different concentrations of Fe3+ (0-6 equiv) in MeOH/water (1/4, v/v) solution. Inset shows the linear responses with Fe3+ concentrations.
In order to investigate the influence of the different acid concentration on the spectra of probe LS1 and found a suitable pH span in which LS1 could selectively
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detect Fe3+ efficiently, the acid titration experiments were performed. As shown in Fig. S6, for free probe LS1, at the condition pH < 5.0, the fluorescence responses of
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F577/F777 enhanced due to the strong protonation of rhodamine moiety. While the pH was between 5.0 and 10.0, no significant fluorescence response change of the free
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LS1 was observed. Besides, in the presence of Fe3+ ions (10 eq.), the pH values, between 5.0 and 10.0, had no effect on the fluorescence intensity ratio at F577/F777. Thus, probe LS1 could be used to detect Fe3+ at a comparatively wide pH rage in the solution of H2O-MeOH (4:1, v/v).
In addition, it was of great interest to investigate the reversibility of probe LS1
for detecting Fe3+ in H2O-MeOH (4:1, v/v) solution. The result was shown in Fig. 6. When excess K3PO4 (20 eq.) was introduced into the solution containing LS1 (10 μM) and Fe3+ (10 eq.), the fluorescence intensity at 577 nm was decreased (the green line) and further addition of 20 equiv. Fe3+ could recover the fluorescence again (the blue line). This observation was assumed to be due to decomplexation of Fe3+ by K3PO4 followed by a spirolactam ring closure reaction. Thus, probe LS1 can be classified as a reversible chemosensor for Fe3+ in the solution of H2O-MeOH (4:1, v/v).
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Fig. 6. Fluorescence intensity of LS1 (10 μM) to Fe3+ in MeOH/water (1/4, v/v) solution. (1) baseline: 10 mM LS1 only; (2): 10 mM LS1 with 10 equiv Fe3+; (3): LS1 with10 equiv. Fe3+ andthen addition of 20 equiv. PO43-; (4): LS1 with 10 equiv. Fe3+ and 20 equiv. PO43+, then addition of 20 equiv. Fe3+. Excitation wavelength: 520 nm, slit: 10 nm. Insert shows the photo of solution A, B, C, D.
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3.2 The detection of Cu2+ in H2O-MeCN solution
The UV-vis characteristics of probe LS1 in a solution of H2O-MeCN (1:1, v/v) have been studied. And we found that probe LS1 can show totally different detecting
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behavior in a solution of H2O-MeCN (1:1, v/v) from in the solution of H2O-MeOH
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(1:4, v/v) which have been discussed before. As shown in Fig.7 and Fig.S7, probe LS1 (10 μM) exhibited obviously selective response to Cu2+ among 19 tested metal ions. When there were no metal ions being added, the solution of free probe LS1 displayed a single strong absorption band at about 628 nm, which is attributed to the absorption of cyanine moiety. When after all the 19 metal ions (100 μM, except Cu2+ of 20 μM) being added separately, only the solution with Cu2+ immediately changed its color from blue to pink which allowing naked-eye detection. In the meantime, a large range of hypochromatic shift (118 nm) in absorption spectra could be observed as the peak decreased at 628 nm and increased at 510 nm, allowing colorimetric detection of Cu2+. As shown in Fig.S7, the solution of free probe LS1 exhibited a relatively high level absorbance intensity ratiometric value, but upon the addition of 2 equiv. of Cu2+, there was prominent decrescence of this value. In the meantime, under the same condition, the addition of the other 10 eq. metal ions had nearly no response to this value.
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Fig. 7. Absorbance spectra of LS1 (10 μM) in the presence of 10 equiv. different metal ions in MeCN/water (1/1, v/v) solution except Cu2+ for 2 eq.
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As shown in Fig. 8, the competitive experiment also confirmed that the background metal ions showed very low interference with the detection of Cu2+ in the solution of H2O-MeCN (1:1, v/v). It was also investigated that the ratiometric
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absorbance signal response of probe LS1 toward Cu2+ in the presence of 10 eq. coexistent anions (NO3-, I-, Cl-, Br-, HPO42-, H2PO4-, SO42-, CO32-, HCO3- and AcO-),
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which revealed that all the tested anions have little interference on the detecting of Cu2+ (Fig. S8). Therefore, these results suggested that probe LS1 has a high
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selectivity for Cu2+ in the presence of these tested metal ions and anions.
Fig. 8. Ratiometric absorbance response (A628/A510) of LS1 (10 μM) upon addition of 2 eq. Cu2+ in the presence of 10 eq. background metal ions in MeCN/water (1/1, v/v) solution.
The detection limit of probe LS1 has also been evaluated. The absorbance 11
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titration profile of probe LS1 (10 μM) with various concentration of Cu2+ was recorded (Fig.9). Upon addition of increasing concentration of Cu2+ ions to the solution, a new absorption band centered at 510 nm appeared while the absorption band centered at 628 nm disappeared, which could be ascribed to the Cu2+ induced spirocyclic-opening reaction. The linear response of the absorbance intensity ratio (A628/A510) toward Cu2+ was obtained in Cu2+ concentration of 0-20 μM, and the
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detection limit of Cu2+ was measured to be 1.019 µM, which was about 20 times
lower than the WHO recommended value for Cu2+ (2.0 mg/L) in drinking water
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(Fig.S9a). A Job’s plot (Fig.S9b) analysis corresponding to 0.67, the concentration ratio indicated the 2:1 stoichiometric ratio between probe LS1 and Cu2+.
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In order to investigate the influence of the different acid concentration on the spectra of probe LS1 and found a suitable pH span in which LS1 could selectively detect Cu2+ efficiently, the acid titration experiments were performed. As shown in
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Fig. S10, for free probe LS1, at the condition pH < 3.0, the absorbance responses of A628/A510 decrease due to the strong protonation of rhodamine moiety. While the pH
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was from 3.0 to 10.0, no significant absorbance response change of the free LS1 was observed. However, the addition of Cu2+ ions (2 eq.) led to the fluorescence enhancement over a quite wide pH range (3-10), which was attributed to the opening
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of rhodamine ring. Consequently, probe LS1 might be used to detect Cu2+ in
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approximate physiological conditions.
Fig. 9. Absorbance spectra of LS1 (10 µM) in H2O-MeCN (1:1, v/v) solutions with various amounts of Cu2+ ions (0-4 eq.).
Moreover, a time course of the absorbance response of LS1 upon addition Cu2+ was shown in Fig 10. The kinetics of absorbance changement at 510 nm and 628 nm 12
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by the new developed ratiometric fluorescent probe LS1 was recorded, and results indicated that the recognizing event could complete in 1 min so that probe LS1 was a
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selectivity and rapidly sensor for Cu2+ over various other metal ions.
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Fig. 10. Kinetics of the ratiometric absorbance response (A628/A510) of LS1 (10 µM) in the presence of 2 eq Cu2+.
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3.3 Bioimaging applications of compound LS1 in SH-SY5Y109 cells. Bioimaging applications of compound LS1 for monitoring of Cu2+ ions in living cells were carried out [50]. First of all, it is important to determine the cytotoxic effect
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of LS1 and Cu2+ and the complex on living SH-SY5Y109 cells
[51]
. The
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well-established MTT assay was adopted to study cytotoxicity of the aforementioned compounds at varying concentrations mentioned in the Materials and Methods section (Fig S11). As shown in Fig S11, the probe LS1 exerted little adverse effect on cell viability, the same is the case when cells were treated with varying concentrations of CuCl2. SH-SY5Y109 cells were incubated with LS1 (10 μM) in natural water for about 30 min at 37 oC, and as shown in Fig.11b, very weak fluorescence of LS1 inside the living SH-SY5Y109 cells was observed. After washing with water two times, 30 μM of Cu2+ were then supplemented to the cells. After incubated at 37 oC for 30 min later, a significant increase in the fluorescence from the intracellular area was observed (Fig.11d). A bright-field transmission image of cells treated with LS1 and Cu2+ confirmed that the cells were viable throughout the imaging experiments (Fig. 11). These results demonstrated that LS1 might be used for detecting Cu2+ in biological samples.
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(b)
(c)
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Fig. 11. Fluorescence images of Cu2+ in SH-SY5Y109 cells with 10 μM solution of LS1 in H2O
for 30 min at 37 oC, bright-field transmission images (a, c) and fluorescence images (b, d) of
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SH-SY5Y109 cells incubated with 0 μM, 30 μM of Cu2+ for 30 min, respectively (λex = 520 nm,
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red channel).
4. Conclusion
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In conclusion, we synthesized and reported an easily available bifunctional and ratiometric NIR absorptive and fluorescent probe LS1 based on rhodamine B and heptamethine indocyanine. This novel probe can extraordinarily detect Fe3+ and Cu2+
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respectively in solvent of MeOH/H2O and MeCN/H2O, and provide a facile method
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for naked-eye detection of these two metal ions separately. Probe LS1 exhibited clearly Fe3+ or Cu2+-induced changes, with high electivity and sensitivity over other metal ions, in the fluorescent and absorbance ratio of two well-separated NIR and VIS peaks. Moreover, it was applied for imaging in SH-SY5Y109 cells to confirm that it can be used as a fluorescent probe for monitoring Cu2+ in living cells. Although the exact effect of solvent system on metal ion selectivity was not clear at present, the theme of the molecular design presented here may help the development of more efficient rhodamine-based chemosensor platforms. Acknowledgments
This work was supported by the National Science Foundation of China (Nos. 21572209, 21442002) and Science-Technology Foundation for Outstanding Young Scientists of Henan Academy of Agricultural Sciences (Grant no. 2013YQ24 and 2016YQ22).
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Development of Near-Infrared Fluorescent Sensors for in Vivo Imaging. J. Am. Chem. Soc. 2012, [23] L. He, W. Lin, Q. Xu, M. Ren, H. Wei and J. Y. Wang, A simple and effective “capping” 4530-4536.
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[25] K. Zheng, W. Lin, W. Huang, X. Guan, D. Cheng and J. Y. Wang, Facile synthesis of a class of aminochromene-aniliniumion conjugated far-red to near-infrared fluorescent dyes for bioimaging. J. Mater. Chem. B, 2015, 3, 871-877. [26] H. Chen, W. Lin, H. Cui and W. Jiang, Development of Unique Xanthene–Cyanine Fused Near-Infrared Fluorescent Fluorophores with Superior Chemical Stability for Biological Fluorescence Imaging. Chem. Eur. J., 2015, 21, 733-745. [27] K. Kiyose, H. Kojima, Y. Urano, T. Nagano, Development of a ratiometric fluorescent zinc ion probe in near-infrared region, based on tricarbocyanine chromophore, J. Am. Chem. Soc. 128 (2006) 6548-6549. [28] Z. Guo, G. H. Kim, A cyanine-based fluorescent sensor for detecting endogenous zinc ions in live cells and organisms. 33 (2012) 7818-7827. [29] B. Tang, L. J. Cui, K. H. Xu, L. L. Tong, G. W. Yang, L. G. An, A sensitive and selective near-infrared fluorescent probe for mercuric ions and its biological imaging applications, ChemBioChem 9 (2008) 1159-1164. 16
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[30] M. Zhu, M. Yuan, X. Liu, J. Xu, J. Lv, C. Huang, H. Liu, Y. Li, S. Wang, D. Zhu, Visible near-infrared chemosensor for mercury ion, Org. Lett. 10 (2008) 1481-1484. [31] Z. Guo, W. Zhu, M. Zhu, X. Wu, H. Tian, Near-infrared cell-permeable Hg2+-selective ratiometric fluorescent chemodosimeters and fast indicator paper for MeHg+ based on tricarbocyanines, Chem. Eur. J. 16 (2010) 14424-14432. [32] H. Zheng, X. J. Zhang, X. Cai, Q. N. Bian, M. Yan, G. H. Wu, X. W. Lai, Y. B. Jiang, Ratiometric fluorescent chemosensor for Hg2+ based on heptamethine cyanine containing a
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thymine moiety, Org. Lett. 14 (2012) 1986-1989.
[33] Y. Yang, T. Cheng, W. Zhu, Y. Xu, X. Qian, Highly selective and sensitive near-infrared
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fluorescent sensors for cadmium in aqueous solution. Org. Lett. 13 (2011) 264-267.
[34] P. Li, X. Duan, Z. Chen, Y. Liu, T. Xie, L. Fang, X. Li, M. Yin, B. Tang, A near-infrared imaging applications, Chem. Commun. 47 (2011) 7755-7757.
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fluorescent probe for detecting copper(II) with high selectivity and sensitivity and its biological [35] H. Zheng, M. Yan, X. X. Fan, D. Sun, S. Y. Yang, L. J. Yang, J. D. Li, Y. B. Jiang, A
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heptamethine cyanine-based colorimetric and ratiometric fluorescent chemosensor for the selective detection of Ag+ in an aqueous medium, Chem. Commun. 48 (2012) 2243-2245. [36] C. Y. Li, X. F. Kong, Y. F. Li, C. X. Zou, D. Liu, W. G. Zhu, Ratiometric and colorimetric
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fluorescent chemosensor for Ag+ based on tricarbocyanine, Dyes Pigm. 99 (2013) 903-907. [37] R. Y. Tsien, A. T. Harootunian, Practical design criteria for a dynamic ratio imaging system, Cell Calcium 11 (1990) 93–109.
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[38] A. Coskun, E. U. Akkaya, Ion sensing coupled to resonance energy transfer: a highly
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selective and sensitive ratiometric fluorescent chemosensor for Ag(I) by a modular approach, J. Am. Chem. Soc. 127 (2005) 10464-10465.
[39] W. Lin, L. Yuan, L. Long, C. Guo, J. Feng, A fluorescent cobalt probe with a large ratiometric fluorescence response via modulation of energy acceptor molar absorptivity on metal ion binding, Adv. Funct. Mater. 18 (2008) 2366-2372.
[40] L. Yuan, W. Lin, B. Chen, Y. Xie, Development of FRET-based ratiometric fluorescent Cu2+ chemodosimeters and the applications for living cell imaging, Org. Lett. 14 (2012) 432-435. [41] J. Fan, M. Hu, P. Zhan, X. Peng, Energy transfer cassettes based on organic fluorophores: construction and applications in ratiometric sensing, Chem. Soc. Rev. 42 (2013) 29-43. [42] X. Ma, Z. Tan, G. Wei, D. Wei and Y. Du, Solvent controlled sugar–rhodamine
fluorescence sensor for Cu2+ detection. Analyst, 137 (2012) 1436-1439. [43] H. J. Jung, N. Singh, D. Y. Lee, D. O. Jang, Single sensor for multiple analytes: chromogenic detection of I- and fluorescent detection of Fe3+. Tetrahedron Letters 51 (2010) 3962-3965. [44] L. Tang, J. Guo and Z. Huang, Tune Metal Ion Selectivity by Changing Working Solvent: Fluorescent and Colorimetric Recognition of Cu2+ by a Known Hg2+ Selective Probe. Bull. Korean Chem. Soc, 34 (2013), 1061-1064. [45] L. Tang, F. Li, M. Liu, R. Nandhakumar, Single sensor for two metal ions: Colorimetric 17
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recognition of Cu2+ and fluorescent recognition of Hg2+. Spectrochimica Acta Part A 78 (2011) 1168-1172. [46] Z. Xu, H. Wang, X. Hou, W. Xu, T. Xiang, C. Wu, A novel ratiometric colorimetric and NIR fluorescent probe for detecting Cu2+ with high selectivity and sensitivity based on rhodamineappended cyanine. Sensors and Actuators B: Chemical, 201 (2014) 469-474. [47] P. Job. Annalidi Chimica 9 (1928) 113 -116. [48] Y. Zhou, J. Zhang, L. Zhang, Q. Zhang, T. Ma, J. Niu, A rhodamine-based fluorescent
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enhancement chemosensor for the detection of Cr3+ in aqueous media. Dyes and Pigments. 97 (2013) 148-154.
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[49] D. Liu, T. Pang, K. Ma, W. Jiang and X. Bao, A new highly sensitive and selective
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conjugate. RSC Adv. 4 (2014) 2563-2567.
[50] C. Wang, D. Zhang, X. Huang, P. Ding, Z. Wang, Y. Zhao, Y. Ye, A ratiometric fluorescent chemosensor for Hg2+ based on FRET and its application in living cells. Sensors and Actuators B:
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[51] S. Goswami, K. Aich, S. Das, C. D. Mukhopadhyay, D. Sarkarc and T. K. Mondal, A new visible-light-excitable ICT-CHEF-mediated fluorescence ‘turn-on’ probe for the selective
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Biographies
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5763-5770.
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detection of Cd2+ in a mixed aqueous system with live-cell imaging. Dalton Trans., 2015, 44,
Shao Li received his BE degree from Xi’an University of Science and Technology, PR China, in 2012. He is currently a master candidate at the College of Chemistry and Molecular Engineering, Zhengzhou University. His research interests focus on fluorescent sensors.
Di Zhang received his PhD degree in 2014 from Zhengzhou University, PR China. He is now working in Institute of Agricultural Quality Standards and Testing Technology, Henan Academy of Agricultural Sciences. His current research interests focus on developing fluorescent probes.
Xinyu Xie is currently an undergraduate at the School of Life Sciences, Zhengzhou University. His research interests focus on fluorescent sensors and chemical biology. 18
Page 18 of 25
Saige Ma received her BS degree from Luoyang Institute of Science and Technology, PR China, in 2014. She is currently a master candidate at the College of Chemistry and Molecular Engineering, Zhengzhou University. Her research interests focus on
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fluorescent sensors.
Yao Liu received his BS degree from Xinyang Normal University, PR China, in 2012.
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He is currently a master candidate at the College of Chemistry and Molecular Engineering, Zhengzhou University. His research interests focus on fluorescent
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sensors.
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Zhanhui Xu received his PhD degree in 2003 from Institute of Chemistry Chinese Academy of Sciences, PR China. He is a lecturer at the College of Chemistry and
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Molecular Engineering, Zhengzhou University. His current research interests include
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development of complex synthesis and phosphorus chemistry.
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Prof. Yanfeng Gao received his PhD degree in 2006 from Lanzhou university, PR China. He is now working in the School of Life Sciences, Zhengzhou University. His current research interests include organic synthesis and chemical biology.
Prof. Yong Ye received his PhD degree in 2003 from Nankai University, PR China. He is a professor at the College of Chemistry and Molecular Engineering, Zhengzhou University. His current research interests include development of complex synthesis, sensors and phosphorus chemistry. 19
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Fig. S1. 1H NMR chart of probe LS1 (CDCl3, 400 MHz).
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Fig. S2. 13C NMR chart of probe LS1 (CDCl3, 100 MHz)
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ls-1 #267 RT: 0.62 AV: 1 NL: 6.96E9 T: FTMS + p ESI Full ms [500.00-1500.00]
931.56671
100 95
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90 85
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80 75 70 Relative Abundance
65 60 55 50 45 40 35 30 25 20 15 10
5
719.45300 0 700
762.44049 788.45288
859.61115
750
850
800
903.53540 900
977.56183 1007.60223 950 m/z
1000
1117.69153 1050
1100
1150
1200
Fig. S3. ESI-HRMS spectrum of probe LS1.
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Fig. S4. Fluorescence spectra of LS1 (10 μM) in the presence of 10 equiv different metal ions in MeOH/water (1/4, v/v) solution. λex = 520 nm, scan range 540–800 nm, slit width 10 nm.
3+
Fig. S5. Fluorescence intensity ratio (F577/F777) of LS1 to Fe containing various anions in MeOH/water (1/4, v/v) solution. [LS1] = 10 µM, [Fe3+ ] = [Anion] = 10 eq, λex = 520 nm, scan range 540–800 nm, slit width 10 nm.
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Fig. S6. Fluorescence intensity ratio (F577/F777) of free LS1 (10 µM) and in the presence of 10 equiv. Fe3+ in NaOH/HCl, MeOH/H2O (1/4, v/v) solution with different pH conditions.
Fig. S7. Ratiometric absorbance response (A628/A510) in the presence of 10 eq. different metal ions in MeCN/water (1/1, v/v) solution (Cu2+ for 2 eq.).
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Fig. S8. Ratiometric absorbance response (A628/A510) of LS1 to Cu2+ containing various anions in MeCN/water (1/1, v/v) solution. [LS1] = 10 µM, [Cu2+ ] = [Anion] = 10 eq.
Fig. S9. (a) linear responses with Cu2+ concentrations. (b) Job's plot for determining the stoichiometry of LS1 and Cu2+, ([LS1] + Cu2+ = 100 mΜ, λ = 510 nm)
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Fig. S10. Ratiometric absorbance response (A628/A510) of free LS1 (10 µM) and in the presence of 2 eq. Cu2+ in MeCN/water (1/1, v/v) solution with different pH (100 mM Tris/HCl) conditions.
Fig. S11. It represents % cell viability of SH-SY5Y109 cells treated with various concentrations (2.5 µM–15 µM) of LS1 for 24 h determined by MTT assay. Results are expressed as mean of three independent experiments.
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S0925-4005(15)30552-9 http://dx.doi.org/doi:10.1016/j.snb.2015.10.086 SNB 19230
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
14-8-2015 17-10-2015 24-10-2015
Please cite this article as: S. Li, D. Zhang, X. Xie, S. Ma, Y. Liu, Z. Xu, Y. Gao, Y. Ye, A novel solvent-dependently bifunctional NIR absorptive and fluorescent ratiometric probe for detecting Fe3+ /Cu2+ and its application in bioimaging, Sensors and Actuators B: Chemical (2015), http://dx.doi.org/10.1016/j.snb.2015.10.086 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A novel solvent-dependently bifunctional NIR absorptive and fluorescent ratiometric probe for detecting Fe3+/Cu2+ and its application in bioimaging
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Shao Lia, Di Zhangb*, Xinyu Xiec, Saige Maa, Yao Liua, Zhanhui Xua, Yanfeng
Phosphorus Chemical Engineering Research Center of Henan Province, the College
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a
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Gaoc, Yong Yea*
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of Chemistry and Molecular Engineering, Zhengzhou University, Zhengzhou 450052, China.
Institute of Agricultural Quality Standards and Testing Technology, Henan Academy
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b
of Agricultural Sciences, Zhengzhou 450002, China.
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The School of Life Sciences, Zhengzhou University, Zhengzhou 450001, China.
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c
∗
Corresponding author; Email: [email protected] (Di Zhang); [email protected] (Yong Ye) 1
Page 1 of 25
Abstract: A novel rhodamine-cyanine based probe LS1 was designed and synthesized, which can act as a bifunctional NIR ratiometric colorimetric and fluorescent probe for detecting Fe3+ and Cu2+ in solvent of MeOH/H2O and MeCN/H2O respectively. As expected, it exhibited high selectivity and sensitivity for detecting Fe3+ and Cu2+ over
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other commonly coexistent metal ions in their respective systems with a broad pH span. The detection limit was measured to be 0.737 µM for Fe3+ and 1.019 µM for
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Cu2+. Furthermore, fluorescence imaging experiments of Cu2+ ions in living SH-SY5Y109 cells demonstrated its value of practical application in biological
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systems.
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Keywords: NIR, fluorescent probe, Fe3+, Cu2+, Cyanine, Rhodamine 1. Introduction Recently, the design and development of chemosensors for sensing and recognition of environmentally and biologically important heavy- and transition-metal ions has attracted wide-spread interests of biologists, chemists, clinical biochemists [1-5]
. Among transition metal ions, iron is the most abundant
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and environmentalists
essential trace element for both plants and animals. Fe3+ plays an important role in
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enzyme catalysis, cellular metabolism, and as an oxygen carrier in hemoglobin and a
cofactor in many enzymatic reactions involved in the mitochondrial respiratory chain [6] [7]
, Parkinson’s disease
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. Both of its deficiency and excess can result in serious disorders such as Huntington [8]
. And copper, rank only to iron and zinc, is the third most
abundant essential trace element in the human body, and performs some important
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roles in many fundamental physiological processes in organisms [9], but excessive levels of cooper have malign influences [10-12]. Therefore, many excellent works of reported and investigated [13-19].
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Fe3+ and Cu2+ sensing by synthesized colorimetric/fluorescent probes have been Most of these chemosensors have the absorption and emission in the
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ultraviolet-visible (UV/Vis) light rage, which renders them difficult to be employed
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for sensing and imaging targets of interest in living lives, as the absorption and auto-fluorescence of biomolecules in UV/Vis region are high [20]. Recently, cyanine dyes, as a long wavelength (NIR region at around 650-900 nm) analyte-responsive fluorescent probes, have become the focus of analytical and biological. As it has minimum photo-damage to biological samples, deep tissue penetration, and minimum interference form background auto-fluorescence by biomolecules in the living systems, cyanine dyes have been well used in vivo fluorescence imaging [21-26]. And as the heptamethine cyanine has a rigid chlorocyclohexenyl ring in the polymethine chain that could increase its photostability, enhance the fluorescence quantum yield, and provide an ideal site for further modification with amino or phenol substitutions, we choose heptamethine cyanine as a fluorophore along with other cyanine analogues. According to our knowledge, only few NIR analyte-responsive probes based on heptamethine cyanine for sensing metal ions have been reported [27-36]. However, in the detecting of most probes, the absorbance and fluorescence intensity measurements may be influenced by the variations in the assay sample 3
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environment (pH, concentration, temperature, and so forth), due to the only optical signal change. To eliminate those effects, a ratiometric colorimetric/fluorescent [37-39]
measurement is desirable
. Because ratiometric chemosensors can provide a
built-in correction for environmental effects by simultaneous measurement of two fluorescence signals at different wavelengths followed by calculation of their intensity ratio
[40-41]
. Although some ratiometric chemosensors have been constructed recently,
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there barely are reports on the ratiometric sensing of metal ions in the near-infrared (NIR) region. Thus, there is still an intense demand for new ratiometric optical probes
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for Fe3+/Cu2+ with favorable photophysical proprieties in the NIR region.
Tuning metal ion selectivity by altering the solvent system is in keep with the
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newly emerged chemosensor design concept of “single sensor for multiple analytes”, namely, analysis of more than one analyte by one receptor using a single or an array of detection method
[42-45]
. In this paper, we reported a novel probe LS1, based on
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cyanine-coupled rhodamine B, that could detect Fe3+ and Cu2+ ions in two kinds of
2. Experimental 2.1 Apparatus
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solvent systems separately with high selectivity and sensitivity.
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Absorption spectra were measured on a on a UV-2102 double-beam UV/VIS
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spectrometer, Perkin Elmer precisely. Fluorescence spectra were recorded on the F-4500 FL Spectrophotometer, and the excitation and emission wavelength band passes were both set at 10.0 nm. The pH was measured with a Model pHs-3C meter (Shanghai, China). 1H and 13C NMR spectra were recorded using a Bruker DTX-400 spectrometer. Samples were dissolved in CDCl3 and placed in 5 mm NMR tubes. TMS was used as internal reference. ESI mass spectra were carried out on an HPLC Q-Exactive HR-MS spectrometer (Thermo, USA) by using methanol as mobile phase. Fluorescence images experiments were carried out with a Nikon-80i inverted fluorescence microscope. 2.2 Materials All chemical reagents were used as received from commercial sources without further purification. Solvents for chemical synthesis and analysis were purified according to standard procedures. Deionized water was used throughout the experiment. Chloride salts of metal ions (Li+, K+, Na+, Ca2+, Mg2+, Ba2+, Zn2+, Fe2+, Mn2+, Cu2+, Co2+, Ni2+, Cd2+, Cr3+, Hg2+, Al3+) and the nitrate salts of Ag+, Pb2+ and 4
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Fe3+ ions were prepared as 10.00 mM in water solution. 2.3 Synthesis N
O
N
N O
N
N Cl
+
H N
N EtOH O
ref lux, 8 h
N NH2
NI
O
N I
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N
LS1
2
1
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Scheme 1. Synthetic route of probe LS1.
Compound 1 and 2 was synthesized by reported methods [46].
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Compound 2 (0.61 g, 1 mM) was dissolved in anhydrous ethanol (20 mL) in a round bottom flask, then compound 1 (0.97 g, 2 mM) was added. The mixture was
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heated to reflux, and stirred under N2 atmosphere for 8 h. Then the solvent of resulting mixture was evaporated in vacuum. The residue was purified by silica gel thin layer chromatography with EtOAc/MeOH (8:1, v/v) to obtain 0.46 g of dark blue 1
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solid in a yield of 43 %.
H NMR (400 MHz, CDCl3, ppm) δ: 1.14 (t, 12 H, J = 7.0 Hz), 1.30 (t, 2 H, J = 13.6
Hz), 1.52 (s, 12 H), 1.67 (t, 2 H, J = 6.0 Hz), 2.41 (bs, 4 H), 2.90 (t, 2 H, J = 14.0 Hz),
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3.08 (s, 1 H), 3.31 (m, 8 H), 3.49 (s, 3 H), 5.19 (d, 2 H, J = 12.4 Hz), 6.18 (d, 1 H, J =
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2.4 Hz), 6.20 (d, 1 H, J = 2.4 Hz), 6.35 (d, 2 H, J = 2.3 Hz), 6.43 (d, 2 H, J = 8.8), 6.57 (d, 2 H, J = 7.8 Hz), 6.80 (t, 2 H, J = 7.4 Hz), 7.10 (m, 6 H), 7.40 (t, 2 H, J = 3.5 Hz), 7.91 (t, 1 H, J = 4.6 Hz);
13
C NMR (100 MHz, CDCl3, ppm) δ: 12.65, 14.12,
21.41, 22.69, 26.33, 28.62, 29.69, 44.34, 44.41, 93.62, 97.87, 97.98, 104.77, 107.49, 108.00, 108.24, 121.63, 121.73, 122.73, 124.08, 127.51, 128.02, 128.28, 128.47, 128.82, 132.92, 139.60, 144.11, 148.88, 148.92, 153.41, 153.77; HR-MS m/z: Calcd for C62H71N6O2+ [M-I-]+ 931.5633, found 931.5667. 3. Results and analysis
Probe LS1 was dissolved in MeCN or MeOH to make a 1 mM stock solution, respectively. Then the stock solution was further diluted to require concentration for measurement. 3.1 The detection of Fe3+ in H2O-MeOH solution. The UV-vis and Fluorescence characteristics of probe LS1 in a solution of H2O-MeOH (4:1, v/v) have been studied. As shown in Fig. 1 and Fig. S4, probe LS1 5
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(10 μM) exhibited obviously selective response to Fe3+ among 19 tested metal ions (Li+, K+, Na+, Ca2+, Mg2+, Ba2+, Zn2+, Fe2+, Mn2+, Pb2+, Cu2+, Ag+, Fe3+, Co2+, Ni2+, Cd2+, Cr3+, Hg2+, Al3+). When there were no metal ions being added, the solution of probe LS1 displayed a single strong absorption band at about 650 nm, which is attributed to the absorption of cyanine moiety. After all the 19 metal ions (100 μM) were added separately into the solution of probe LS1 and placed for 30 min, only the
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solution containing Fe3+ changed its color from blue to red obviously which allowing
naked-eye detection. In the meantime, a large range of hypochromatic shift (136 nm)
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increased at 514 nm, allowing colorimetric detection of Fe3+.
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in absorption spectrum could be observed as the peak decreased at 650 nm and
Fig. 1. Absorbance spectra of LS1 (10 μM) in the absence and presence of 10 eq. different metal ions in MeOH/water (1/4, v/v) solution. Inset shows the photo of LS1with different metal ions.
The free probe LS1 exhibited an emission band at about 777 nm attributed to
cyanine moiety and another emission band at about 577 nm attributed to rhodamine moiety (Fig. S4). A prominent enhancement of characteristic fluorescence of rhodamine B emerged at 577 nm after 10 eq. of Fe3+ was added into the solution. Meanwhile, under the same condition, no obvious response could be observed upon the addition of the same amount of other ions. A mild increase of fluorescence at 577 nm was also detected when the same amount of Al3+ was added due to their low binding affinity to probe LS1. It was noteworthy that the difference between the two emission wavelength was quite large (almost 200 nm), which not only contribute to the accurate measurement of the intensities of these two emission peaks, but also resulted in a large ratiometric value. As shown in Fig. 2, the solution of free probe LS1 exhibited a very low fluorescence intensity ratiometric value, but upon the 6
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addition of 10 eq. of Fe3+, there was prominent enhancement of this value. In the meantime, under the same condition, the addition of the other ions had almost no response to this value, except a mild fluorescence enhancement factor was also detected for Al3+. Furthermore, as shown in Fig. 3, the competitive experiment also confirmed that the background metal ions showed very low interference with the detection of Fe3+ in
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the solution of H2O-MeOH (4:1, v/v). It was also investigated that the ratiometric fluorescence signal response of probe LS1 toward Fe3+ in the presence of various
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coexistent anions such as NO3-, I-, Cl-, Br-, H2PO4-, SO42-, HCO3- and AcO-, which revealed that all the tested anions have little interference on the detecting of Fe3+
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(Fig.S5). Therefore, these results suggested that probe LS1 has a high selectivity for
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Fe3+ in the presence of these tested foreign metal ions and anions.
Fig. 2. Fluorescence intensity ratio (F577/F777) of LS1 (10 μM) in the presence of 10 equiv different metal ions in MeOH/water (1/4, v/v) solution. λex = 520 nm, scan range 540–800 nm, slit width 10 nm.
Fig. 3. Fluorescence intensity ratio (F577/F777) of LS1 (10 μM) upon addition of 10 equiv Fe3+in the presence of 10 equiv background metal ions in MeOH/water (1/4, v/v) solution. 7
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To further investigate the interaction between Fe3+ and probe LS1, an ultraviolet tritration experiment was carried out (Fig. 4). Upon addition of increasing concentration of Fe3+ ions to the solution, a new absorption band centered at 514 nm appeared while the absorption band centered at 650 nm disappeared, which could be ascribed to the Fe3+ induced spirocyclic-pening reaction accompanied with activating
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the photo-induced electron transfer (PET) process of cyanine to Fe3+ metal ion. A Job’s plot (Fig. 4 inset) analysis corresponding to 0.50 the concentration ratio
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indicated the 1:1 stoichiometric ratio between probe LS1 and Fe3+.[47]
Fig. 4. Absorption spectra of LS1 (10 µM) with gradual addition of various amounts of Fe3+ (from bottom 0-6 eq) in MeOH/water (1/4, v/v) solution. Inset shows Job's plot for determining the stoichiometry of LS1 and Fe3+, ([LS1] + [Fe3+] = 100 μM).
To investigate the practical applicability of probe LS1, the detection limit of this
new chemosensor was evaluated. The fluorescence titration profile of probe LS1 (10 μM) with various concentration of Fe3+ was recorded at excitation wavelength of 520 nm (Fig. 5). The linear response of the emission intensity ratio (F577/F777) toward Fe3+ was obtained in Fe3+ concentration of 0-20 μM, and the detection limit (3σ/slope) of probe LS1 for the determination of Fe3+ was found to be 0.737 µM (Fig.5. inset) [48-49]
.
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Fig. 5. Fluorescence emission spectra of compound LS1 (10 μM) in the presence of different concentrations of Fe3+ (0-6 equiv) in MeOH/water (1/4, v/v) solution. Inset shows the linear responses with Fe3+ concentrations.
In order to investigate the influence of the different acid concentration on the spectra of probe LS1 and found a suitable pH span in which LS1 could selectively
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detect Fe3+ efficiently, the acid titration experiments were performed. As shown in Fig. S6, for free probe LS1, at the condition pH < 5.0, the fluorescence responses of
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F577/F777 enhanced due to the strong protonation of rhodamine moiety. While the pH was between 5.0 and 10.0, no significant fluorescence response change of the free
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LS1 was observed. Besides, in the presence of Fe3+ ions (10 eq.), the pH values, between 5.0 and 10.0, had no effect on the fluorescence intensity ratio at F577/F777. Thus, probe LS1 could be used to detect Fe3+ at a comparatively wide pH rage in the solution of H2O-MeOH (4:1, v/v).
In addition, it was of great interest to investigate the reversibility of probe LS1
for detecting Fe3+ in H2O-MeOH (4:1, v/v) solution. The result was shown in Fig. 6. When excess K3PO4 (20 eq.) was introduced into the solution containing LS1 (10 μM) and Fe3+ (10 eq.), the fluorescence intensity at 577 nm was decreased (the green line) and further addition of 20 equiv. Fe3+ could recover the fluorescence again (the blue line). This observation was assumed to be due to decomplexation of Fe3+ by K3PO4 followed by a spirolactam ring closure reaction. Thus, probe LS1 can be classified as a reversible chemosensor for Fe3+ in the solution of H2O-MeOH (4:1, v/v).
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Fig. 6. Fluorescence intensity of LS1 (10 μM) to Fe3+ in MeOH/water (1/4, v/v) solution. (1) baseline: 10 mM LS1 only; (2): 10 mM LS1 with 10 equiv Fe3+; (3): LS1 with10 equiv. Fe3+ andthen addition of 20 equiv. PO43-; (4): LS1 with 10 equiv. Fe3+ and 20 equiv. PO43+, then addition of 20 equiv. Fe3+. Excitation wavelength: 520 nm, slit: 10 nm. Insert shows the photo of solution A, B, C, D.
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3.2 The detection of Cu2+ in H2O-MeCN solution
The UV-vis characteristics of probe LS1 in a solution of H2O-MeCN (1:1, v/v) have been studied. And we found that probe LS1 can show totally different detecting
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behavior in a solution of H2O-MeCN (1:1, v/v) from in the solution of H2O-MeOH
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(1:4, v/v) which have been discussed before. As shown in Fig.7 and Fig.S7, probe LS1 (10 μM) exhibited obviously selective response to Cu2+ among 19 tested metal ions. When there were no metal ions being added, the solution of free probe LS1 displayed a single strong absorption band at about 628 nm, which is attributed to the absorption of cyanine moiety. When after all the 19 metal ions (100 μM, except Cu2+ of 20 μM) being added separately, only the solution with Cu2+ immediately changed its color from blue to pink which allowing naked-eye detection. In the meantime, a large range of hypochromatic shift (118 nm) in absorption spectra could be observed as the peak decreased at 628 nm and increased at 510 nm, allowing colorimetric detection of Cu2+. As shown in Fig.S7, the solution of free probe LS1 exhibited a relatively high level absorbance intensity ratiometric value, but upon the addition of 2 equiv. of Cu2+, there was prominent decrescence of this value. In the meantime, under the same condition, the addition of the other 10 eq. metal ions had nearly no response to this value.
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Fig. 7. Absorbance spectra of LS1 (10 μM) in the presence of 10 equiv. different metal ions in MeCN/water (1/1, v/v) solution except Cu2+ for 2 eq.
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As shown in Fig. 8, the competitive experiment also confirmed that the background metal ions showed very low interference with the detection of Cu2+ in the solution of H2O-MeCN (1:1, v/v). It was also investigated that the ratiometric
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absorbance signal response of probe LS1 toward Cu2+ in the presence of 10 eq. coexistent anions (NO3-, I-, Cl-, Br-, HPO42-, H2PO4-, SO42-, CO32-, HCO3- and AcO-),
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which revealed that all the tested anions have little interference on the detecting of Cu2+ (Fig. S8). Therefore, these results suggested that probe LS1 has a high
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selectivity for Cu2+ in the presence of these tested metal ions and anions.
Fig. 8. Ratiometric absorbance response (A628/A510) of LS1 (10 μM) upon addition of 2 eq. Cu2+ in the presence of 10 eq. background metal ions in MeCN/water (1/1, v/v) solution.
The detection limit of probe LS1 has also been evaluated. The absorbance 11
Page 11 of 25
titration profile of probe LS1 (10 μM) with various concentration of Cu2+ was recorded (Fig.9). Upon addition of increasing concentration of Cu2+ ions to the solution, a new absorption band centered at 510 nm appeared while the absorption band centered at 628 nm disappeared, which could be ascribed to the Cu2+ induced spirocyclic-opening reaction. The linear response of the absorbance intensity ratio (A628/A510) toward Cu2+ was obtained in Cu2+ concentration of 0-20 μM, and the
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detection limit of Cu2+ was measured to be 1.019 µM, which was about 20 times
lower than the WHO recommended value for Cu2+ (2.0 mg/L) in drinking water
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(Fig.S9a). A Job’s plot (Fig.S9b) analysis corresponding to 0.67, the concentration ratio indicated the 2:1 stoichiometric ratio between probe LS1 and Cu2+.
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In order to investigate the influence of the different acid concentration on the spectra of probe LS1 and found a suitable pH span in which LS1 could selectively detect Cu2+ efficiently, the acid titration experiments were performed. As shown in
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Fig. S10, for free probe LS1, at the condition pH < 3.0, the absorbance responses of A628/A510 decrease due to the strong protonation of rhodamine moiety. While the pH
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was from 3.0 to 10.0, no significant absorbance response change of the free LS1 was observed. However, the addition of Cu2+ ions (2 eq.) led to the fluorescence enhancement over a quite wide pH range (3-10), which was attributed to the opening
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of rhodamine ring. Consequently, probe LS1 might be used to detect Cu2+ in
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approximate physiological conditions.
Fig. 9. Absorbance spectra of LS1 (10 µM) in H2O-MeCN (1:1, v/v) solutions with various amounts of Cu2+ ions (0-4 eq.).
Moreover, a time course of the absorbance response of LS1 upon addition Cu2+ was shown in Fig 10. The kinetics of absorbance changement at 510 nm and 628 nm 12
Page 12 of 25
by the new developed ratiometric fluorescent probe LS1 was recorded, and results indicated that the recognizing event could complete in 1 min so that probe LS1 was a
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selectivity and rapidly sensor for Cu2+ over various other metal ions.
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Fig. 10. Kinetics of the ratiometric absorbance response (A628/A510) of LS1 (10 µM) in the presence of 2 eq Cu2+.
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3.3 Bioimaging applications of compound LS1 in SH-SY5Y109 cells. Bioimaging applications of compound LS1 for monitoring of Cu2+ ions in living cells were carried out [50]. First of all, it is important to determine the cytotoxic effect
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of LS1 and Cu2+ and the complex on living SH-SY5Y109 cells
[51]
. The
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well-established MTT assay was adopted to study cytotoxicity of the aforementioned compounds at varying concentrations mentioned in the Materials and Methods section (Fig S11). As shown in Fig S11, the probe LS1 exerted little adverse effect on cell viability, the same is the case when cells were treated with varying concentrations of CuCl2. SH-SY5Y109 cells were incubated with LS1 (10 μM) in natural water for about 30 min at 37 oC, and as shown in Fig.11b, very weak fluorescence of LS1 inside the living SH-SY5Y109 cells was observed. After washing with water two times, 30 μM of Cu2+ were then supplemented to the cells. After incubated at 37 oC for 30 min later, a significant increase in the fluorescence from the intracellular area was observed (Fig.11d). A bright-field transmission image of cells treated with LS1 and Cu2+ confirmed that the cells were viable throughout the imaging experiments (Fig. 11). These results demonstrated that LS1 might be used for detecting Cu2+ in biological samples.
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(b)
(c)
(d)
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(a)
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Fig. 11. Fluorescence images of Cu2+ in SH-SY5Y109 cells with 10 μM solution of LS1 in H2O
for 30 min at 37 oC, bright-field transmission images (a, c) and fluorescence images (b, d) of
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SH-SY5Y109 cells incubated with 0 μM, 30 μM of Cu2+ for 30 min, respectively (λex = 520 nm,
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red channel).
4. Conclusion
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In conclusion, we synthesized and reported an easily available bifunctional and ratiometric NIR absorptive and fluorescent probe LS1 based on rhodamine B and heptamethine indocyanine. This novel probe can extraordinarily detect Fe3+ and Cu2+
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respectively in solvent of MeOH/H2O and MeCN/H2O, and provide a facile method
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for naked-eye detection of these two metal ions separately. Probe LS1 exhibited clearly Fe3+ or Cu2+-induced changes, with high electivity and sensitivity over other metal ions, in the fluorescent and absorbance ratio of two well-separated NIR and VIS peaks. Moreover, it was applied for imaging in SH-SY5Y109 cells to confirm that it can be used as a fluorescent probe for monitoring Cu2+ in living cells. Although the exact effect of solvent system on metal ion selectivity was not clear at present, the theme of the molecular design presented here may help the development of more efficient rhodamine-based chemosensor platforms. Acknowledgments
This work was supported by the National Science Foundation of China (Nos. 21572209, 21442002) and Science-Technology Foundation for Outstanding Young Scientists of Henan Academy of Agricultural Sciences (Grant no. 2013YQ24 and 2016YQ22).
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fluorescence sensor for Cu2+ detection. Analyst, 137 (2012) 1436-1439. [43] H. J. Jung, N. Singh, D. Y. Lee, D. O. Jang, Single sensor for multiple analytes: chromogenic detection of I- and fluorescent detection of Fe3+. Tetrahedron Letters 51 (2010) 3962-3965. [44] L. Tang, J. Guo and Z. Huang, Tune Metal Ion Selectivity by Changing Working Solvent: Fluorescent and Colorimetric Recognition of Cu2+ by a Known Hg2+ Selective Probe. Bull. Korean Chem. Soc, 34 (2013), 1061-1064. [45] L. Tang, F. Li, M. Liu, R. Nandhakumar, Single sensor for two metal ions: Colorimetric 17
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recognition of Cu2+ and fluorescent recognition of Hg2+. Spectrochimica Acta Part A 78 (2011) 1168-1172. [46] Z. Xu, H. Wang, X. Hou, W. Xu, T. Xiang, C. Wu, A novel ratiometric colorimetric and NIR fluorescent probe for detecting Cu2+ with high selectivity and sensitivity based on rhodamineappended cyanine. Sensors and Actuators B: Chemical, 201 (2014) 469-474. [47] P. Job. Annalidi Chimica 9 (1928) 113 -116. [48] Y. Zhou, J. Zhang, L. Zhang, Q. Zhang, T. Ma, J. Niu, A rhodamine-based fluorescent
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[51] S. Goswami, K. Aich, S. Das, C. D. Mukhopadhyay, D. Sarkarc and T. K. Mondal, A new visible-light-excitable ICT-CHEF-mediated fluorescence ‘turn-on’ probe for the selective
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Biographies
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5763-5770.
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detection of Cd2+ in a mixed aqueous system with live-cell imaging. Dalton Trans., 2015, 44,
Shao Li received his BE degree from Xi’an University of Science and Technology, PR China, in 2012. He is currently a master candidate at the College of Chemistry and Molecular Engineering, Zhengzhou University. His research interests focus on fluorescent sensors.
Di Zhang received his PhD degree in 2014 from Zhengzhou University, PR China. He is now working in Institute of Agricultural Quality Standards and Testing Technology, Henan Academy of Agricultural Sciences. His current research interests focus on developing fluorescent probes.
Xinyu Xie is currently an undergraduate at the School of Life Sciences, Zhengzhou University. His research interests focus on fluorescent sensors and chemical biology. 18
Page 18 of 25
Saige Ma received her BS degree from Luoyang Institute of Science and Technology, PR China, in 2014. She is currently a master candidate at the College of Chemistry and Molecular Engineering, Zhengzhou University. Her research interests focus on
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fluorescent sensors.
Yao Liu received his BS degree from Xinyang Normal University, PR China, in 2012.
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He is currently a master candidate at the College of Chemistry and Molecular Engineering, Zhengzhou University. His research interests focus on fluorescent
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sensors.
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Zhanhui Xu received his PhD degree in 2003 from Institute of Chemistry Chinese Academy of Sciences, PR China. He is a lecturer at the College of Chemistry and
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Molecular Engineering, Zhengzhou University. His current research interests include
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development of complex synthesis and phosphorus chemistry.
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Prof. Yanfeng Gao received his PhD degree in 2006 from Lanzhou university, PR China. He is now working in the School of Life Sciences, Zhengzhou University. His current research interests include organic synthesis and chemical biology.
Prof. Yong Ye received his PhD degree in 2003 from Nankai University, PR China. He is a professor at the College of Chemistry and Molecular Engineering, Zhengzhou University. His current research interests include development of complex synthesis, sensors and phosphorus chemistry. 19
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an
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Fig. S1. 1H NMR chart of probe LS1 (CDCl3, 400 MHz).
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Fig. S2. 13C NMR chart of probe LS1 (CDCl3, 100 MHz)
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ls-1 #267 RT: 0.62 AV: 1 NL: 6.96E9 T: FTMS + p ESI Full ms [500.00-1500.00]
931.56671
100 95
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90 85
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80 75 70 Relative Abundance
65 60 55 50 45 40 35 30 25 20 15 10
5
719.45300 0 700
762.44049 788.45288
859.61115
750
850
800
903.53540 900
977.56183 1007.60223 950 m/z
1000
1117.69153 1050
1100
1150
1200
Fig. S3. ESI-HRMS spectrum of probe LS1.
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Fig. S4. Fluorescence spectra of LS1 (10 μM) in the presence of 10 equiv different metal ions in MeOH/water (1/4, v/v) solution. λex = 520 nm, scan range 540–800 nm, slit width 10 nm.
3+
Fig. S5. Fluorescence intensity ratio (F577/F777) of LS1 to Fe containing various anions in MeOH/water (1/4, v/v) solution. [LS1] = 10 µM, [Fe3+ ] = [Anion] = 10 eq, λex = 520 nm, scan range 540–800 nm, slit width 10 nm.
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Fig. S6. Fluorescence intensity ratio (F577/F777) of free LS1 (10 µM) and in the presence of 10 equiv. Fe3+ in NaOH/HCl, MeOH/H2O (1/4, v/v) solution with different pH conditions.
Fig. S7. Ratiometric absorbance response (A628/A510) in the presence of 10 eq. different metal ions in MeCN/water (1/1, v/v) solution (Cu2+ for 2 eq.).
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Fig. S8. Ratiometric absorbance response (A628/A510) of LS1 to Cu2+ containing various anions in MeCN/water (1/1, v/v) solution. [LS1] = 10 µM, [Cu2+ ] = [Anion] = 10 eq.
Fig. S9. (a) linear responses with Cu2+ concentrations. (b) Job's plot for determining the stoichiometry of LS1 and Cu2+, ([LS1] + Cu2+ = 100 mΜ, λ = 510 nm)
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Fig. S10. Ratiometric absorbance response (A628/A510) of free LS1 (10 µM) and in the presence of 2 eq. Cu2+ in MeCN/water (1/1, v/v) solution with different pH (100 mM Tris/HCl) conditions.
Fig. S11. It represents % cell viability of SH-SY5Y109 cells treated with various concentrations (2.5 µM–15 µM) of LS1 for 24 h determined by MTT assay. Results are expressed as mean of three independent experiments.
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