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An oxalamidoquinoline-based fluorescent sensor for selective detection of Zn2+ in solution and living cells and its logic gate behavior
Accepted Manuscript Title: An oxalamidoquinoline-based fluorescent sensor for selective detection of Zn2+ in solution and living cells and its logic gate behavior Author: Xiujuan Tian Xiangfeng Guo Fashuo Yu Lihua Jia PII: DOI: Reference:
S0925-4005(16)30420-8 http://dx.doi.org/doi:10.1016/j.snb.2016.03.126 SNB 19931
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
20-1-2016 20-3-2016 23-3-2016
Please cite this article as: Xiujuan Tian, Xiangfeng Guo, Fashuo Yu, Lihua Jia, An oxalamidoquinoline-based fluorescent sensor for selective detection of Zn2+ in solution and living cells and its logic gate behavior, Sensors and Actuators B: Chemical http://dx.doi.org/10.1016/j.snb.2016.03.126 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.
An oxalamidoquinoline-based fluorescent sensor for selective detection of Zn2+ in solution and living cells and its logic gate behavior
Xiujuan Tian, Xiangfeng Guo*, Fashuo Yu, Lihua Jia*
College of Chemistry and Chemical Engineering, Key Laboratory of Fine Chemicals of College of Heilongjiang Province, Qiqihar University, Qiqihar 161006, China.
*Corresponding authors. Email: [email protected] (Jia L.), [email protected] (Guo X.).
Graphical Abstract
350 nm
350 nm
Zn2+
N O
NH
O
N H
N O
O
OH
490 nm
O
N
-
N-
Zn2+ O
OH
Highlights A new fluorescent sensor based on 8-oxalamidoquinoline (OAQ) has been synthesized. OAQ could recognize Zn2+ without interference from Cd2+. OAQ could be applied in fluorescence imaging in yeast cells. The fluorescence signal of OAQ could act as INHIBIT logic gate by two chemical inputs.
Abstract: A novel fluorescent sensor for Zn2+ based on 8-oxalamidoquinoline (OAQ) was designed and synthesized. OAQ behaves high selectivity to Zn2+ accompanied by remarkable emission enhancement (32 folds) at 490 nm upon binding Zn2+ in buffer aqueous solution at pH 7.2. The results show that a 1:1 complex was formed and the apparent association constant of OAQ/Zn2+ complex was calculated to be 2.5 × 105 M-1. The detection limit was gained to be 2.0 × 10-8 M. Furthermore, the fluorescent signals of OAQ are utilized to construct an INHIBIT logic gate at the molecular level. Additionally OAQ was successfully used for fluorescence imaging in living yeast cells.
Keywords: fluorescence; sensor; oxalamidoquinoline; zinc ion; cell imaging; logic gate 1. Introduction As the second most abundant transition-metal ion in human body [1, 2], zinc ion plays an important part in many biological processes and the metabolic imbalance of Zn2+ will cause many diseases [3-6]. It is necessary to develop effective method to detect Zn2+. As we all know, both Zn2+ and Cd2+ are group 12 elements and possess a closed-shell d10 configuration, which leads to similar properties of them [2, 7, 8]. Therefore, the change of fluorescence intensity as well as the shift of wavelengths is usually observed similar when Zn2+ and Cd2+ coordinated with sensors [9-12]. Accordingly, it is a rigorous challenge to detect Zn2+ without the influences of Cd2+ [13-15]. So far, large numbers of fluorescent sensors for selective detection of Zn2+ have been reported [16-19]. Because of the advantages of strong coordination ability, favorable aqueous solubility, excellent biocompatibility, 8-carboxamidoquinoline-based sensors [20-24] have attracted serious concern of chemists and our group has also made great effort to study it [25-29]. It is worth mentioning that the sensors are mostly obtained with substituting the chlorine of 8-chloroacetyl quinolone by oxygen or nitrogen [30-34]. We wondered if the oxalamidoquinoline derivatives have similar properties to the above-mentioned sensors. Considering the excellent coordination ability as well as the diversification of structure
(symmetric and asymmetric, cis and trans), oxamide derivatives are ideal ligand for metal ions [35-37] and widely applied in electrode modified [38], anticancer [39], gelators [40] and other aspects. Tzeng [41] et al. reported some gold (I) complexes of oxamide derivatives and their supramolecular assembly. Schröder [42] et al. developed an organic framework based on oxamide-Cu2+, which could be responsible for selective uptake of CO2. However, there have been few reports about oxamide derivatives used as fluorescence sensors to detect metal ions up to now. On the other hand, fluorescent molecular logic gates (making fluorescent signal as output and external factors, such as biomolecules, pH, anions and cations, as input [43-46]) have attracted the attention of chemists since the first fluorescent molecular logic gate has been reported by de Silva [47] et al. In these systems, the output, ‘‘on” or ‘‘off’’ switching of fluorescence signal, was represented by ‘‘1’’ or ‘‘0’’ and it is in response to the addition ‘‘1’’ or no addition ‘‘0’’ of the input chemicals. Taking all above into consideration, we designed and synthesized a simple, easily available as well as water-soluble fluorescent sensor based on 8-oxalamidoquinoline (OAQ), which is able to distinguish zinc ions from cadmium ions by the changes of both absorbance and emission spectra of OAQ. Another, OAQ was successfully used for fluorescence imaging in yeast cells. It was also found that the fluorescence signal of OAQ could act as an INHIBIT logic gate operated by two chemical inputs Zn2+ (Input1) and Cu2+ or Ni2+ (Input2). 2. Experimental 2.1. Materials and methods All solvents were obtained from commercial suppliers (analytical grade) and used as received without further purification. Water was purified by Milli-Q purification system. ESI-MS spectra were measured with Agilent 6310 ESI-Ion Trop Mass spectrometer. NMR spectra were recorded on a Bruker Avance-600 spectrometer and referenced to internal tetramethylsilane. FT-IR spectra were measured with Nicolet Avatar-370. Melting points were got on X-6 micro melting point apparatus. Fluorescence steady state spectra and UV-vis absorption spectra were recorded on a Hitachi F-7000 and Puxi TU-1901 spectrophotometer, respectively. All pH measurements were made using a Sartorius basic pH-meter PB-10. Imaging experiments were performed on Olympus BX51 fluorescence microscope with
excitation at 330-385 nm and a DP71 color CCD camera was used to collect photos. The eyepiece and objective lens were set as 10× and 100×, respectively. 2.2. General procedure for spectroscopic measurements All of the detections of metal ions were operated at pH 7.2 maintained with Tris-HClO4 buffer (0.01 M). The metal ion solutions (0.050 M) were obtained from NaCl, KCl, Mg(ClO4)2, Ca(NO3)2, Cr(NO3)2, FeSO4, CoSO4, NiSO4, Cu(NO3)2, Zn(NO3)2, AgNO3, CdSO4, HgCl2, Pb(NO3)2, and AlCl3. All fluorescence data were recorded with the excitation of 350 nm, and the slit widths of excitation and emission were 2.5 nm and 5.0 nm, respectively. 2.3. Synthesis of OAQ The structure and synthetic route of the sensor are shown in Scheme 1 and all characterization results of EOQ, as well as OAQ, were given in supporting information.
2.3.1. Preparation of ethyl 2-oxo-2-(quinolin-8-ylamino)acetate (EOQ). 8-Aminoquinoline (1.01 g, 6.98 mmol) and diethyl oxalate (12 mL) were mixed and refluxed for 3 hours. The crude products were purified by column chromatography (DCM, Rf = 0.80) and then evaporated the solvent under reduced pressure to afford a white solid called EOQ (1.34 g). Yield: 92.0 %, Mp: 76.8–77.3 oC. 1H NMR (600 MHz, CDCl3) δ (ppm): 11.39 (s, 1H), 8.90 (t, J = 1.8 Hz, 1H), 8.80 (d, J = 7.2 Hz, 1H), 8.20 (d, J = 8.4 Hz, 1H), 7.57–7.63 (m, 2H), 7.50 (q, J = 4.2 Hz, 1H), 4.49 (q, J = 7.2 Hz, 2H), 1.48 (t, J = 7.2 Hz, 3H). 13C NMR (150 MHz, CDCl3) δ (ppm): 160.7, 154.2, 148.9, 138.7, 136.2, 133.0, 127.9, 127.1, 123.2, 121.9, 117.3, 63.5 and 14.0. FTIR (KBr) v (cm-1): 3320, 2980, 1530, 1490, 1290 and 1160. 2.3.2. Preparation of N-(2-(2-hydroxyethoxy)ethyl)-N’-(quinolin-8-yl)oxalamide (OAQ). EOQ (550 mg, 2.25 mmol) and 2-(2-aminoethoxy) ethanol (357 mg, 3.38 mmol) were added to ethanol (10 mL), after refluxed for 2 hours, the mixture was cooled to room temperature and the solvent was removed under reduced pressure to obtain a white solid, which was purified by silica gel column chromatography using ethyl acetate as eluent to afford OAQ (607 mg). Yield: 88.9%. 1H NMR (600 MHz, CDCl3) δ (ppm): 11.64 (s, 1H), 8.90 (dd, J = 1.8, 4.2 Hz, 1H), 8.73 (dd, J = 1.8, 7.2 Hz, 1H), 8.17 (dd, J = 1.8, 8.4 Hz, 1H), 7.93 (s, 1H) , 7.60 (m, 2H), 7.48 (q, J = 4.2 Hz, 1H), 7.43 (s, 1H), 3.78 (t, J = 4.2 Hz, 2H),
3.69 (m, 2H), 3.64 (m, 4H), 2.11 (br, 1H). 13C NMR (150 MHz, CDCl3) δ (ppm): 160.1, 157.8, 149.0, 139.0, 136.1, 133.0, 128.0, 126.9, 123.1, 121.9, 117.1, 72.4, 69.8, 61.8 and 39.8. FTIR (KBr) v (cm-1): 3477, 3309, 2888, 1658, 1484, 1328, 1126 and 1079. MS (ESI): m/z calculated for [C15H17N3O4 + H]+: 304.1297, found 304.1467. 2.4. Cell imaging experiments After being dispersed in YPD medium (1.4 % yeast extract, 4.0 % peptone, 2.0 % glucose, 0.2 % (NH4)2SO4) and incubated for 24 h at 37 oC, yeast was used as a cell model. For yeast staining, some colonies were selected and incubated with 0.10 mM OAQ in Tris-HClO4 (0.01 M) solution (pH 7.2) for 2 h at 37 oC. After removing the sensor solution and ringing twice with PBS buffer (0.01 M, pH 7.4), the cells pretreated by OAQ were diluted with the solution of ZnSO4 (40 μM) and incubated for another 2 h at 37 oC. Then the cells for experiments were viewed under a fluorescent microscope as well as bright field. 3. Results and Discussion 3.1 Metal ions sensing studies of OAQ The fluorescence and absorbance spectra of OAQ were investigated in methanol-water Tris-HClO4 buffer solution. As shown in Fig. 1a, when excited with 350 nm, an emission peak was found around 400 nm which maybe result from the Raman scattering of solvent, since their wavelengths shifted when the excited wavelength were changed (Fig. S8 see ESI). In addition, the fluorescence emission around 490 nm was not apparent and the spectra were not obviously affected by the most of common metal ions including Na+, K+, Mg2+, Ca2+, Cr3+, Fe2+, Co2+, Ni2+, Cu2+, Ag+, Cd2+, Hg2+, Pb2+, and Al3+. While a 32-fold fluorescence enhancement of OAQ at 490 nm was observed when Zn2+ was added, which mainly resulted from the coordinating OAQ to Zn2+ and alone with the deprotonation of the amide group. [25, 27]. Furthermore, the effect of Zn2+ and Cd2+ on absorption spectra of OAQ was compared. As is shown in Fig. 1b, a significant red-shift of the absorption spectrum was observed after 5 equiv. Zn2+ added, while the changes of the absorbance spectrum could be ignored when 5 equiv. Cd2+ added. The combination of OAQ and Zn2+ was accompanied by deprotonation process of 8-amide group, and the deprotonation process strengthened the electron-donating ability from the nitrogen atom of the 8-amide group to the quinoline ring. Then the electron transfer from the nitrogen atom of the heterocycle to the metal ion further enhanced the ICT
process [25]. As a result, a red-shift in absorption wavelength could be observed. Taking the changes of both fluorescence and absorbance spectra into consideration, OAQ showed high selectivity for Zn2+ over other metal ions, and particularly the interference of Cd2+could avoid. To further gauging the anti-disturbance performance of OAQ for Zn2+ over other metal ions, the competition experiments were carried by adding 5 equiv. Zn2+ to the solutions of OAQ (1.0 × 10−5 M) in the presence of 5 equiv. other metal ions, and the results were shown in Fig. 2. When 5.0 equiv. Zn2+ was added to the solutions containing the same amount of other metal ions (Na+, K+, Mg2+, Ca2+, Cr3+, Fe2+, Co2+, Ni2+, Cu2+, Ag+, Cd2+, Hg2+, Pb2+, and Al3+), there were notable fluorescence increasing being observed except Ni2+ or Cu2+ solution added. As we all known, Cu2+ and Ni2+ are paramagnetic ions with empty d-shell and after bonding with sensors, the emission will be strongly quenched via a photoinduced metal to fluorophore electron or energy transfer mechanism [48]. In addition, Co2+ could induce a slight disturbance on emission intensity increase. In brief, sensor OAQ for Zn2+ detection had satisfactory anti-disturbance performance over other metal ions.
The titration experiments were implemented in the presence of different concentrations of Zn2+ and the results were showed in Fig. 3. From the absorbance titration profile (Fig. 3a), it was found that the addition of Zn2+ led to the increasing of absorbance at 246, 292 and 347 nm. At the same time, four well-defined isobestic points were observed at 239, 268, 300 and 320 nm, respectively, which implied the coexistence of OAQ and OAQ–Zn2+ complex. From the inset of Fig. 3a, the rapidly increasing of absorbance at 347 nm was discovered upon gradual addition of Zn2+ (from 0 to 10 μM), and then the increasing got slowly with the concentration of Zn2+ ranged from 10 to 30 μM. At last the saturated spectra were obtained. Meanwhile, as the concentration of Zn2+ increased, with λex = 350 nm, the fluorescence peak at 490 nm increased (Fig. 3b). Upon increasing the concentration of Zn2+, a similar result was observed (compared with the absorbance at 347 nm). The fluorescence emission at 490 nm increased linearly with the Zn2+ concentration ranged from 0 to 9 μM and the linear equation was found to be Y = 5.561 + 4.510X (linearly dependent coefficient: R2 = 0.9916), just as shown in Fig. 4. The limit of detection (LOD) was evaluated to be 2.0 × 10-8 M with 3σ / slope [49, 50]. Also the binding constant of OAQ with Zn2+ was got from fluorescence
titration data. As shown in Fig. 5, the binding constant which was obtained by the Benesi– Hildebrand equation [51, 52] was 2.5 × 105 M-1.
To determine the binding stoichiometry of OAQ–Zn2+ complex, the Job’s plot was adapted. As shown in Fig. 6, the Job’s plot, by using the changes of fluorescence intensity at 490 nm as a function of molar fraction of Zn2+, showed that the maximum of If 490 nm obtained at the molar fraction of Zn2+ being 0.5. That is to say, the binding stoichiometry for Zn2+ and OAQ is 1:1. This also had been proved by the ESI MS study (Fig. 7). The [OAQ + Zn2+ – H+]+ complex was calculated at m/z 366.0427 and measured at m/z 366.0604.
Fluorescent chemosensors are usually disturbed by proton in the detection of metal ions [53]. In order to eliminate the disturbance by protonation or deprotonation of the fluorophore during the detection, the effect of pH on the fluorescence signal of OAQ in the absence and presence of Zn2+ was studied. As shown in Fig. 8, along with the increase of pH from 2.0 to 4.6, the fluorescence intensity of 490 nm was almost overlapping, which may be due to the protonation of the quinoline nitrogen atom and the inhibition of the deprotonation of amido group in strong acid solution. In other words, the combination between OAQ and Zn2+ was very weak in the acidic environment. Then it became quite different. From pH 4.6 to 6.8, rapidly increasing in fluorescence emission of OAQ was found with the presence of 5 equiv. Zn2+. Then satisfactory binding ability of OAQ to Zn2+ was exhibited when the pH located between 6.8 and 7.7. Subsequently, it gradually decreased with pH continuing increased. The maximum value of If 490 nm was reached at pH 7.2 and it was chosen as the test condition, which is consistent with the physiological pH value.
3.2. Bioimaging applications of OAQ in live yeast cells To investigate the potential biological application of OAQ in living cells, the intracellular Zn2+ imaging of yeast cells was performed by fluorescence microscopy. As shown in Fig. 9, after incubation with DMSO-containing OAQ (0.10 mM) for 2 h at 37 oC, cells never showed fluorescence. While, blue fluorescence became visible when exogenous Zn2+ (40 μM) was introduced. That is to say, OAQ has the potential for live cell imaging and can be used in
detection of Zn2+ in live cells.
3.3. Logic gate behavior of OAQ The fluorescence behavior of OAQ at 490 nm mimics the INHIBIT logic gate, the truth table and the circuit for the INHIBIT logic gate are shown in Table 1 and Fig. 10, respectively. The fluorescence output values (If 490 nm) below the predefined threshold level are correspond to binary ‘‘0’’, while the output values above the threshold translated into binary ‘‘1’’. Just as shown in Fig. 10. (a), the fluorescence emission of OAQ (If 490 nm) was wake (‘‘0’’ state, bar a). When Zn2+ ion was present, the fluorescence became strong (‘‘1’’ state, bar b). While in the presence of Cu2+ (or Ni2+) ion If 490 nm was observed to be low (‘‘0’’ state, bar c). When both Zn2+ and Cu2+ (or Ni2+) ions were added, system still exhibited a low fluorescence (‘‘0’’ state, bar d). This behavior was well consistent with INHIBIT logic gates.
Conclusions
In summary, we designed and synthesized an oxalamidoquinoline derivative, OAQ, which showed a selective fluorescence enhancement against Zn2+ and the effect of Cd2+ could be ignored. An approximately 32-fold Zn2+ selective fluorescence enhancement response in Tris-HClO4 (10 mM, at pH 7.2), CH3OH-H2O (1:9, v/v) was obtained and the 1:1 complex was proved by both the Job’s plot and MS spectrum study. Additionally, the fluorescence behavior of OAQ was well consistent with INHIBIT logic gates. Moreover, OAQ could also detect Zn2+ of yeast cells. Therefore, OAQ will satisfy the criteria for further biological and environmental applications according to the ideal chemical, biological and spectroscopic properties.
Acknowledgments This work was supported by the National Natural Science Foundation of China (21176125), and the Research Foundation of Education Bureau of Heilongjiang Province of China (2012TD012).
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Biographies Xiujuan Tian received her B.S. degree from Langfang Teachers University, PR China, in 2013. She is currently a master candidate at College of Chemistry and Chemical Engineering, Qiqihar University, PR China. Her research interests focus on molecular recognition. Xiangfeng Guo received his Ph.D. degree from Dalian University of Technology, PR China, in 2004. He is a professor in College of Chemistry and Chemical Engineering, Qiqihar University. His current research interests are mainly in the development of chemosensors and surfactants. Fashuo Yu received his B.S. degree from Dalian Polytechnic University, PR China, in 2014. He is currently a master candidate at College of Chemistry and Chemical Engineering, Qiqihar University, PR China. His research interests focus on developing fluorescent chemosensors. Lihua Jia received her Ph.D. degree from Dalian University of Technology, PR China, in 2004. She is a professor in College of Chemistry and Chemical Engineering, Qiqihar University. Her current research interests include supramolecular chemistry and the development of catalyst.
Figure Captions Fig. 1. Fluorescence (a) and absorbance (b) spectra of 10 μM OAQ in the absence and presence of different metal ions (5 equiv.) at pH 7.2 (10 mM Tris-HClO4, CH3OH/H2O = 1:9 (v/v), λex = 350 nm). (Inset) Visible emission observed from OAQ in the absence and presence of Zn2+ (5 equiv) irradiated with 365 nm- UV lamp. Fig. 2. Fluorescence response of OAQ (10 μM) to various tested metal ions (50 μM) (black bar) and to the mixture of 50 μM of tested metal ions with 50 μM Zn2+ ion (gray bar). (λex = 350 nm, λem = 490 nm, CH3OH/H2O = 1:9, v/v, pH = 7.2, 10 mM Tris-HClO4) Fig. 3. Absorbance (a) and fluorescence (b) spectra of probe 10 μM OAQ with Zn2+ (0–11.2 equiv.) at pH 7.2 (10 mM Tris-HClO4, CH3OH/H2O = 1:9 (v/v), λex = 350 nm). The inset shows the absorbance and fluorescence intensity of OAQ as a function of Zn2+ concentration. Fig. 4. Curve of fluorescence intensity at 490 nm of OAQ (10 μM) versus increasing concentrations of Zn2+ at pH 7.2 (10 mM Tris–HClO4, CH3OH/H2O = 1:9 (v/v), λex = 350 nm) Fig. 5. Benesi–Hildebrand plot from fluorescence titration data of OAQ (10 μM) at pH 7.2. (10 mM Tris–HClO4, CH3OH/H2O = 1:9 (v/v), λex = 350 nm) Fig. 6. A Job’s plot for OAQ (forms 1:1 complexes) at pH 7.2 (10 mM Tris–HClO4, CH3OH/H2O = 1:9 (v/v)). The total [OAQ] + [Zn2+] = 10 μM Fig. 7. HRMS spectrum of OAQ in the presence of Zn2+. Fig. 8. Fluorescence intensity of OAQ (10 μM) at various pH values (CH3OH/H2O = 1:9, v/v) in the absence and presence of Zn2+ (5 equiv.). Fig. 9. Fluorescence images of yeast cells. Bright-field (a, c) and fluorescence (b, d) microscopy images of yeast cells incubated for 2 h with OAQ (0.10 mM), without (a, b) and with (c, d) the addition of Zn2+ (40 μM). Fig. 10. Operation of the INHIBIT logic gate. (a) The fluorescence intensities of OAQ at λem = 490 nm in the presence of different inputs. (b) INHIBIT logic scheme.
Scheme 1 Synthesis route of sensors
Table. 1 Truth table for the INHIBIT logic gate.
Fig. 1
2.0
(a) 2+
Zn
75
(b)
1.5
Absorbance
Fluorescence Intensity (a.u.)
100
50
OAQ 2+ OAQ + Zn 2+ OAQ + Cd
1.0
0.5
25
None and others
0 360
0.0
420
480 540 600 Wavelength (nm)
660
Fig. 2
90
OAQ + M
n+
OAQ + M
n+
+ Zn
2+
If 490 nm
60
30
2+
2+
2+
2+
3+
2+
Metal Ions
Cr Fe Co Ni Cu
3+
+
2+
Pb Al Ca
No ne Na + K+ M g 2+ Ag + Cd 2 + Hg 2
0
200
250 300 350 Wavelength (nm)
400
Fig. 3
0.4
0.08 0.04 0.00 0
0.2
239 nm
40 80 2+ [Zn ]/M
320 nm
120
2+
[Zn ]
268 nm 300 nm
0.0 200
250
300 350 Wavelength (nm)
400
Fig. 4 50 Y = 5.561 + 4.510X 2 R = 0.9916
40
If 490 nm
30 20 10 0 0
2
4 6 2+ [Zn ]/M
8
10
(b)
80
2+
[Zn ]
If 490 nm
A 347 nm
Absorbance
100
0.12
(a)
Fluorescence Intensity (a.u.)
0.6
80 40 0 0
60
40 80 120 2+ [Zn ]/M
40 20 0 360
420
480 540 600 Wavelength (nm)
660
Fig. 5
0.06 Y = 0.01148+4.573E-8X 2 R = 0.9877
1/(I - I0)
0.05 0.04 0.03 0.02 0.01 0
300000
600000 2+ 1/[Zn ]
900000
Fig. 6 25
If 490 nm
20 15 10 5 0
0.0
0.2
0.4 0.6 0.8 Zinc molar fraction
1.0
Fig. 7
Fig. 8
100
OAQ 2+ OAQ + Zn
If 490 nm
80 60 40 20 0
2
4
6 pH
8
10
12
Fig. 9
Fig. 10 80
(a)
If 490 nm
60
40 ON/OFF Threshld
20
0
a
b
c
d
Scheme 1
N NH2
O
HO
(COOC2H5)2 Reflux HN
N OEt
NH2
EtOH, Reflux
O O
O O EOQ
8-AQ
N NH N H
Table 1
Input1
Input 2
Output
Zn2+
Cu2+ (or Ni2+)
If 490 nm
0
0
0
1
0
1
0
1
0
1
1
0
OH O OAQ
S0925-4005(16)30420-8 http://dx.doi.org/doi:10.1016/j.snb.2016.03.126 SNB 19931
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
20-1-2016 20-3-2016 23-3-2016
Please cite this article as: Xiujuan Tian, Xiangfeng Guo, Fashuo Yu, Lihua Jia, An oxalamidoquinoline-based fluorescent sensor for selective detection of Zn2+ in solution and living cells and its logic gate behavior, Sensors and Actuators B: Chemical http://dx.doi.org/10.1016/j.snb.2016.03.126 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.
An oxalamidoquinoline-based fluorescent sensor for selective detection of Zn2+ in solution and living cells and its logic gate behavior
Xiujuan Tian, Xiangfeng Guo*, Fashuo Yu, Lihua Jia*
College of Chemistry and Chemical Engineering, Key Laboratory of Fine Chemicals of College of Heilongjiang Province, Qiqihar University, Qiqihar 161006, China.
*Corresponding authors. Email: [email protected] (Jia L.), [email protected] (Guo X.).
Graphical Abstract
350 nm
350 nm
Zn2+
N O
NH
O
N H
N O
O
OH
490 nm
O
N
-
N-
Zn2+ O
OH
Highlights A new fluorescent sensor based on 8-oxalamidoquinoline (OAQ) has been synthesized. OAQ could recognize Zn2+ without interference from Cd2+. OAQ could be applied in fluorescence imaging in yeast cells. The fluorescence signal of OAQ could act as INHIBIT logic gate by two chemical inputs.
Abstract: A novel fluorescent sensor for Zn2+ based on 8-oxalamidoquinoline (OAQ) was designed and synthesized. OAQ behaves high selectivity to Zn2+ accompanied by remarkable emission enhancement (32 folds) at 490 nm upon binding Zn2+ in buffer aqueous solution at pH 7.2. The results show that a 1:1 complex was formed and the apparent association constant of OAQ/Zn2+ complex was calculated to be 2.5 × 105 M-1. The detection limit was gained to be 2.0 × 10-8 M. Furthermore, the fluorescent signals of OAQ are utilized to construct an INHIBIT logic gate at the molecular level. Additionally OAQ was successfully used for fluorescence imaging in living yeast cells.
Keywords: fluorescence; sensor; oxalamidoquinoline; zinc ion; cell imaging; logic gate 1. Introduction As the second most abundant transition-metal ion in human body [1, 2], zinc ion plays an important part in many biological processes and the metabolic imbalance of Zn2+ will cause many diseases [3-6]. It is necessary to develop effective method to detect Zn2+. As we all know, both Zn2+ and Cd2+ are group 12 elements and possess a closed-shell d10 configuration, which leads to similar properties of them [2, 7, 8]. Therefore, the change of fluorescence intensity as well as the shift of wavelengths is usually observed similar when Zn2+ and Cd2+ coordinated with sensors [9-12]. Accordingly, it is a rigorous challenge to detect Zn2+ without the influences of Cd2+ [13-15]. So far, large numbers of fluorescent sensors for selective detection of Zn2+ have been reported [16-19]. Because of the advantages of strong coordination ability, favorable aqueous solubility, excellent biocompatibility, 8-carboxamidoquinoline-based sensors [20-24] have attracted serious concern of chemists and our group has also made great effort to study it [25-29]. It is worth mentioning that the sensors are mostly obtained with substituting the chlorine of 8-chloroacetyl quinolone by oxygen or nitrogen [30-34]. We wondered if the oxalamidoquinoline derivatives have similar properties to the above-mentioned sensors. Considering the excellent coordination ability as well as the diversification of structure
(symmetric and asymmetric, cis and trans), oxamide derivatives are ideal ligand for metal ions [35-37] and widely applied in electrode modified [38], anticancer [39], gelators [40] and other aspects. Tzeng [41] et al. reported some gold (I) complexes of oxamide derivatives and their supramolecular assembly. Schröder [42] et al. developed an organic framework based on oxamide-Cu2+, which could be responsible for selective uptake of CO2. However, there have been few reports about oxamide derivatives used as fluorescence sensors to detect metal ions up to now. On the other hand, fluorescent molecular logic gates (making fluorescent signal as output and external factors, such as biomolecules, pH, anions and cations, as input [43-46]) have attracted the attention of chemists since the first fluorescent molecular logic gate has been reported by de Silva [47] et al. In these systems, the output, ‘‘on” or ‘‘off’’ switching of fluorescence signal, was represented by ‘‘1’’ or ‘‘0’’ and it is in response to the addition ‘‘1’’ or no addition ‘‘0’’ of the input chemicals. Taking all above into consideration, we designed and synthesized a simple, easily available as well as water-soluble fluorescent sensor based on 8-oxalamidoquinoline (OAQ), which is able to distinguish zinc ions from cadmium ions by the changes of both absorbance and emission spectra of OAQ. Another, OAQ was successfully used for fluorescence imaging in yeast cells. It was also found that the fluorescence signal of OAQ could act as an INHIBIT logic gate operated by two chemical inputs Zn2+ (Input1) and Cu2+ or Ni2+ (Input2). 2. Experimental 2.1. Materials and methods All solvents were obtained from commercial suppliers (analytical grade) and used as received without further purification. Water was purified by Milli-Q purification system. ESI-MS spectra were measured with Agilent 6310 ESI-Ion Trop Mass spectrometer. NMR spectra were recorded on a Bruker Avance-600 spectrometer and referenced to internal tetramethylsilane. FT-IR spectra were measured with Nicolet Avatar-370. Melting points were got on X-6 micro melting point apparatus. Fluorescence steady state spectra and UV-vis absorption spectra were recorded on a Hitachi F-7000 and Puxi TU-1901 spectrophotometer, respectively. All pH measurements were made using a Sartorius basic pH-meter PB-10. Imaging experiments were performed on Olympus BX51 fluorescence microscope with
excitation at 330-385 nm and a DP71 color CCD camera was used to collect photos. The eyepiece and objective lens were set as 10× and 100×, respectively. 2.2. General procedure for spectroscopic measurements All of the detections of metal ions were operated at pH 7.2 maintained with Tris-HClO4 buffer (0.01 M). The metal ion solutions (0.050 M) were obtained from NaCl, KCl, Mg(ClO4)2, Ca(NO3)2, Cr(NO3)2, FeSO4, CoSO4, NiSO4, Cu(NO3)2, Zn(NO3)2, AgNO3, CdSO4, HgCl2, Pb(NO3)2, and AlCl3. All fluorescence data were recorded with the excitation of 350 nm, and the slit widths of excitation and emission were 2.5 nm and 5.0 nm, respectively. 2.3. Synthesis of OAQ The structure and synthetic route of the sensor are shown in Scheme 1 and all characterization results of EOQ, as well as OAQ, were given in supporting information.
2.3.1. Preparation of ethyl 2-oxo-2-(quinolin-8-ylamino)acetate (EOQ). 8-Aminoquinoline (1.01 g, 6.98 mmol) and diethyl oxalate (12 mL) were mixed and refluxed for 3 hours. The crude products were purified by column chromatography (DCM, Rf = 0.80) and then evaporated the solvent under reduced pressure to afford a white solid called EOQ (1.34 g). Yield: 92.0 %, Mp: 76.8–77.3 oC. 1H NMR (600 MHz, CDCl3) δ (ppm): 11.39 (s, 1H), 8.90 (t, J = 1.8 Hz, 1H), 8.80 (d, J = 7.2 Hz, 1H), 8.20 (d, J = 8.4 Hz, 1H), 7.57–7.63 (m, 2H), 7.50 (q, J = 4.2 Hz, 1H), 4.49 (q, J = 7.2 Hz, 2H), 1.48 (t, J = 7.2 Hz, 3H). 13C NMR (150 MHz, CDCl3) δ (ppm): 160.7, 154.2, 148.9, 138.7, 136.2, 133.0, 127.9, 127.1, 123.2, 121.9, 117.3, 63.5 and 14.0. FTIR (KBr) v (cm-1): 3320, 2980, 1530, 1490, 1290 and 1160. 2.3.2. Preparation of N-(2-(2-hydroxyethoxy)ethyl)-N’-(quinolin-8-yl)oxalamide (OAQ). EOQ (550 mg, 2.25 mmol) and 2-(2-aminoethoxy) ethanol (357 mg, 3.38 mmol) were added to ethanol (10 mL), after refluxed for 2 hours, the mixture was cooled to room temperature and the solvent was removed under reduced pressure to obtain a white solid, which was purified by silica gel column chromatography using ethyl acetate as eluent to afford OAQ (607 mg). Yield: 88.9%. 1H NMR (600 MHz, CDCl3) δ (ppm): 11.64 (s, 1H), 8.90 (dd, J = 1.8, 4.2 Hz, 1H), 8.73 (dd, J = 1.8, 7.2 Hz, 1H), 8.17 (dd, J = 1.8, 8.4 Hz, 1H), 7.93 (s, 1H) , 7.60 (m, 2H), 7.48 (q, J = 4.2 Hz, 1H), 7.43 (s, 1H), 3.78 (t, J = 4.2 Hz, 2H),
3.69 (m, 2H), 3.64 (m, 4H), 2.11 (br, 1H). 13C NMR (150 MHz, CDCl3) δ (ppm): 160.1, 157.8, 149.0, 139.0, 136.1, 133.0, 128.0, 126.9, 123.1, 121.9, 117.1, 72.4, 69.8, 61.8 and 39.8. FTIR (KBr) v (cm-1): 3477, 3309, 2888, 1658, 1484, 1328, 1126 and 1079. MS (ESI): m/z calculated for [C15H17N3O4 + H]+: 304.1297, found 304.1467. 2.4. Cell imaging experiments After being dispersed in YPD medium (1.4 % yeast extract, 4.0 % peptone, 2.0 % glucose, 0.2 % (NH4)2SO4) and incubated for 24 h at 37 oC, yeast was used as a cell model. For yeast staining, some colonies were selected and incubated with 0.10 mM OAQ in Tris-HClO4 (0.01 M) solution (pH 7.2) for 2 h at 37 oC. After removing the sensor solution and ringing twice with PBS buffer (0.01 M, pH 7.4), the cells pretreated by OAQ were diluted with the solution of ZnSO4 (40 μM) and incubated for another 2 h at 37 oC. Then the cells for experiments were viewed under a fluorescent microscope as well as bright field. 3. Results and Discussion 3.1 Metal ions sensing studies of OAQ The fluorescence and absorbance spectra of OAQ were investigated in methanol-water Tris-HClO4 buffer solution. As shown in Fig. 1a, when excited with 350 nm, an emission peak was found around 400 nm which maybe result from the Raman scattering of solvent, since their wavelengths shifted when the excited wavelength were changed (Fig. S8 see ESI). In addition, the fluorescence emission around 490 nm was not apparent and the spectra were not obviously affected by the most of common metal ions including Na+, K+, Mg2+, Ca2+, Cr3+, Fe2+, Co2+, Ni2+, Cu2+, Ag+, Cd2+, Hg2+, Pb2+, and Al3+. While a 32-fold fluorescence enhancement of OAQ at 490 nm was observed when Zn2+ was added, which mainly resulted from the coordinating OAQ to Zn2+ and alone with the deprotonation of the amide group. [25, 27]. Furthermore, the effect of Zn2+ and Cd2+ on absorption spectra of OAQ was compared. As is shown in Fig. 1b, a significant red-shift of the absorption spectrum was observed after 5 equiv. Zn2+ added, while the changes of the absorbance spectrum could be ignored when 5 equiv. Cd2+ added. The combination of OAQ and Zn2+ was accompanied by deprotonation process of 8-amide group, and the deprotonation process strengthened the electron-donating ability from the nitrogen atom of the 8-amide group to the quinoline ring. Then the electron transfer from the nitrogen atom of the heterocycle to the metal ion further enhanced the ICT
process [25]. As a result, a red-shift in absorption wavelength could be observed. Taking the changes of both fluorescence and absorbance spectra into consideration, OAQ showed high selectivity for Zn2+ over other metal ions, and particularly the interference of Cd2+could avoid. To further gauging the anti-disturbance performance of OAQ for Zn2+ over other metal ions, the competition experiments were carried by adding 5 equiv. Zn2+ to the solutions of OAQ (1.0 × 10−5 M) in the presence of 5 equiv. other metal ions, and the results were shown in Fig. 2. When 5.0 equiv. Zn2+ was added to the solutions containing the same amount of other metal ions (Na+, K+, Mg2+, Ca2+, Cr3+, Fe2+, Co2+, Ni2+, Cu2+, Ag+, Cd2+, Hg2+, Pb2+, and Al3+), there were notable fluorescence increasing being observed except Ni2+ or Cu2+ solution added. As we all known, Cu2+ and Ni2+ are paramagnetic ions with empty d-shell and after bonding with sensors, the emission will be strongly quenched via a photoinduced metal to fluorophore electron or energy transfer mechanism [48]. In addition, Co2+ could induce a slight disturbance on emission intensity increase. In brief, sensor OAQ for Zn2+ detection had satisfactory anti-disturbance performance over other metal ions.
The titration experiments were implemented in the presence of different concentrations of Zn2+ and the results were showed in Fig. 3. From the absorbance titration profile (Fig. 3a), it was found that the addition of Zn2+ led to the increasing of absorbance at 246, 292 and 347 nm. At the same time, four well-defined isobestic points were observed at 239, 268, 300 and 320 nm, respectively, which implied the coexistence of OAQ and OAQ–Zn2+ complex. From the inset of Fig. 3a, the rapidly increasing of absorbance at 347 nm was discovered upon gradual addition of Zn2+ (from 0 to 10 μM), and then the increasing got slowly with the concentration of Zn2+ ranged from 10 to 30 μM. At last the saturated spectra were obtained. Meanwhile, as the concentration of Zn2+ increased, with λex = 350 nm, the fluorescence peak at 490 nm increased (Fig. 3b). Upon increasing the concentration of Zn2+, a similar result was observed (compared with the absorbance at 347 nm). The fluorescence emission at 490 nm increased linearly with the Zn2+ concentration ranged from 0 to 9 μM and the linear equation was found to be Y = 5.561 + 4.510X (linearly dependent coefficient: R2 = 0.9916), just as shown in Fig. 4. The limit of detection (LOD) was evaluated to be 2.0 × 10-8 M with 3σ / slope [49, 50]. Also the binding constant of OAQ with Zn2+ was got from fluorescence
titration data. As shown in Fig. 5, the binding constant which was obtained by the Benesi– Hildebrand equation [51, 52] was 2.5 × 105 M-1.
To determine the binding stoichiometry of OAQ–Zn2+ complex, the Job’s plot was adapted. As shown in Fig. 6, the Job’s plot, by using the changes of fluorescence intensity at 490 nm as a function of molar fraction of Zn2+, showed that the maximum of If 490 nm obtained at the molar fraction of Zn2+ being 0.5. That is to say, the binding stoichiometry for Zn2+ and OAQ is 1:1. This also had been proved by the ESI MS study (Fig. 7). The [OAQ + Zn2+ – H+]+ complex was calculated at m/z 366.0427 and measured at m/z 366.0604.
Fluorescent chemosensors are usually disturbed by proton in the detection of metal ions [53]. In order to eliminate the disturbance by protonation or deprotonation of the fluorophore during the detection, the effect of pH on the fluorescence signal of OAQ in the absence and presence of Zn2+ was studied. As shown in Fig. 8, along with the increase of pH from 2.0 to 4.6, the fluorescence intensity of 490 nm was almost overlapping, which may be due to the protonation of the quinoline nitrogen atom and the inhibition of the deprotonation of amido group in strong acid solution. In other words, the combination between OAQ and Zn2+ was very weak in the acidic environment. Then it became quite different. From pH 4.6 to 6.8, rapidly increasing in fluorescence emission of OAQ was found with the presence of 5 equiv. Zn2+. Then satisfactory binding ability of OAQ to Zn2+ was exhibited when the pH located between 6.8 and 7.7. Subsequently, it gradually decreased with pH continuing increased. The maximum value of If 490 nm was reached at pH 7.2 and it was chosen as the test condition, which is consistent with the physiological pH value.
3.2. Bioimaging applications of OAQ in live yeast cells To investigate the potential biological application of OAQ in living cells, the intracellular Zn2+ imaging of yeast cells was performed by fluorescence microscopy. As shown in Fig. 9, after incubation with DMSO-containing OAQ (0.10 mM) for 2 h at 37 oC, cells never showed fluorescence. While, blue fluorescence became visible when exogenous Zn2+ (40 μM) was introduced. That is to say, OAQ has the potential for live cell imaging and can be used in
detection of Zn2+ in live cells.
3.3. Logic gate behavior of OAQ The fluorescence behavior of OAQ at 490 nm mimics the INHIBIT logic gate, the truth table and the circuit for the INHIBIT logic gate are shown in Table 1 and Fig. 10, respectively. The fluorescence output values (If 490 nm) below the predefined threshold level are correspond to binary ‘‘0’’, while the output values above the threshold translated into binary ‘‘1’’. Just as shown in Fig. 10. (a), the fluorescence emission of OAQ (If 490 nm) was wake (‘‘0’’ state, bar a). When Zn2+ ion was present, the fluorescence became strong (‘‘1’’ state, bar b). While in the presence of Cu2+ (or Ni2+) ion If 490 nm was observed to be low (‘‘0’’ state, bar c). When both Zn2+ and Cu2+ (or Ni2+) ions were added, system still exhibited a low fluorescence (‘‘0’’ state, bar d). This behavior was well consistent with INHIBIT logic gates.
Conclusions
In summary, we designed and synthesized an oxalamidoquinoline derivative, OAQ, which showed a selective fluorescence enhancement against Zn2+ and the effect of Cd2+ could be ignored. An approximately 32-fold Zn2+ selective fluorescence enhancement response in Tris-HClO4 (10 mM, at pH 7.2), CH3OH-H2O (1:9, v/v) was obtained and the 1:1 complex was proved by both the Job’s plot and MS spectrum study. Additionally, the fluorescence behavior of OAQ was well consistent with INHIBIT logic gates. Moreover, OAQ could also detect Zn2+ of yeast cells. Therefore, OAQ will satisfy the criteria for further biological and environmental applications according to the ideal chemical, biological and spectroscopic properties.
Acknowledgments This work was supported by the National Natural Science Foundation of China (21176125), and the Research Foundation of Education Bureau of Heilongjiang Province of China (2012TD012).
References
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Biographies Xiujuan Tian received her B.S. degree from Langfang Teachers University, PR China, in 2013. She is currently a master candidate at College of Chemistry and Chemical Engineering, Qiqihar University, PR China. Her research interests focus on molecular recognition. Xiangfeng Guo received his Ph.D. degree from Dalian University of Technology, PR China, in 2004. He is a professor in College of Chemistry and Chemical Engineering, Qiqihar University. His current research interests are mainly in the development of chemosensors and surfactants. Fashuo Yu received his B.S. degree from Dalian Polytechnic University, PR China, in 2014. He is currently a master candidate at College of Chemistry and Chemical Engineering, Qiqihar University, PR China. His research interests focus on developing fluorescent chemosensors. Lihua Jia received her Ph.D. degree from Dalian University of Technology, PR China, in 2004. She is a professor in College of Chemistry and Chemical Engineering, Qiqihar University. Her current research interests include supramolecular chemistry and the development of catalyst.
Figure Captions Fig. 1. Fluorescence (a) and absorbance (b) spectra of 10 μM OAQ in the absence and presence of different metal ions (5 equiv.) at pH 7.2 (10 mM Tris-HClO4, CH3OH/H2O = 1:9 (v/v), λex = 350 nm). (Inset) Visible emission observed from OAQ in the absence and presence of Zn2+ (5 equiv) irradiated with 365 nm- UV lamp. Fig. 2. Fluorescence response of OAQ (10 μM) to various tested metal ions (50 μM) (black bar) and to the mixture of 50 μM of tested metal ions with 50 μM Zn2+ ion (gray bar). (λex = 350 nm, λem = 490 nm, CH3OH/H2O = 1:9, v/v, pH = 7.2, 10 mM Tris-HClO4) Fig. 3. Absorbance (a) and fluorescence (b) spectra of probe 10 μM OAQ with Zn2+ (0–11.2 equiv.) at pH 7.2 (10 mM Tris-HClO4, CH3OH/H2O = 1:9 (v/v), λex = 350 nm). The inset shows the absorbance and fluorescence intensity of OAQ as a function of Zn2+ concentration. Fig. 4. Curve of fluorescence intensity at 490 nm of OAQ (10 μM) versus increasing concentrations of Zn2+ at pH 7.2 (10 mM Tris–HClO4, CH3OH/H2O = 1:9 (v/v), λex = 350 nm) Fig. 5. Benesi–Hildebrand plot from fluorescence titration data of OAQ (10 μM) at pH 7.2. (10 mM Tris–HClO4, CH3OH/H2O = 1:9 (v/v), λex = 350 nm) Fig. 6. A Job’s plot for OAQ (forms 1:1 complexes) at pH 7.2 (10 mM Tris–HClO4, CH3OH/H2O = 1:9 (v/v)). The total [OAQ] + [Zn2+] = 10 μM Fig. 7. HRMS spectrum of OAQ in the presence of Zn2+. Fig. 8. Fluorescence intensity of OAQ (10 μM) at various pH values (CH3OH/H2O = 1:9, v/v) in the absence and presence of Zn2+ (5 equiv.). Fig. 9. Fluorescence images of yeast cells. Bright-field (a, c) and fluorescence (b, d) microscopy images of yeast cells incubated for 2 h with OAQ (0.10 mM), without (a, b) and with (c, d) the addition of Zn2+ (40 μM). Fig. 10. Operation of the INHIBIT logic gate. (a) The fluorescence intensities of OAQ at λem = 490 nm in the presence of different inputs. (b) INHIBIT logic scheme.
Scheme 1 Synthesis route of sensors
Table. 1 Truth table for the INHIBIT logic gate.
Fig. 1
2.0
(a) 2+
Zn
75
(b)
1.5
Absorbance
Fluorescence Intensity (a.u.)
100
50
OAQ 2+ OAQ + Zn 2+ OAQ + Cd
1.0
0.5
25
None and others
0 360
0.0
420
480 540 600 Wavelength (nm)
660
Fig. 2
90
OAQ + M
n+
OAQ + M
n+
+ Zn
2+
If 490 nm
60
30
2+
2+
2+
2+
3+
2+
Metal Ions
Cr Fe Co Ni Cu
3+
+
2+
Pb Al Ca
No ne Na + K+ M g 2+ Ag + Cd 2 + Hg 2
0
200
250 300 350 Wavelength (nm)
400
Fig. 3
0.4
0.08 0.04 0.00 0
0.2
239 nm
40 80 2+ [Zn ]/M
320 nm
120
2+
[Zn ]
268 nm 300 nm
0.0 200
250
300 350 Wavelength (nm)
400
Fig. 4 50 Y = 5.561 + 4.510X 2 R = 0.9916
40
If 490 nm
30 20 10 0 0
2
4 6 2+ [Zn ]/M
8
10
(b)
80
2+
[Zn ]
If 490 nm
A 347 nm
Absorbance
100
0.12
(a)
Fluorescence Intensity (a.u.)
0.6
80 40 0 0
60
40 80 120 2+ [Zn ]/M
40 20 0 360
420
480 540 600 Wavelength (nm)
660
Fig. 5
0.06 Y = 0.01148+4.573E-8X 2 R = 0.9877
1/(I - I0)
0.05 0.04 0.03 0.02 0.01 0
300000
600000 2+ 1/[Zn ]
900000
Fig. 6 25
If 490 nm
20 15 10 5 0
0.0
0.2
0.4 0.6 0.8 Zinc molar fraction
1.0
Fig. 7
Fig. 8
100
OAQ 2+ OAQ + Zn
If 490 nm
80 60 40 20 0
2
4
6 pH
8
10
12
Fig. 9
Fig. 10 80
(a)
If 490 nm
60
40 ON/OFF Threshld
20
0
a
b
c
d
Scheme 1
N NH2
O
HO
(COOC2H5)2 Reflux HN
N OEt
NH2
EtOH, Reflux
O O
O O EOQ
8-AQ
N NH N H
Table 1
Input1
Input 2
Output
Zn2+
Cu2+ (or Ni2+)
If 490 nm
0
0
0
1
0
1
0
1
0
1
1
0
OH O OAQ