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A colorimetric and fluorescent probe for multiple transition metal ions (Cu2+, Zn2+ and Ni2+): Fast response and high selectivity
Accepted Manuscript Title: A Colorimetric and Fluorescent Probe for Multiple Transition Metal Ions (Cu2+ , Zn2+ and Ni2+ ): Fast Response and High Selectivity Author: Chunrong Chen Guangwen Men Wenhuan Bu Chunshuang Liang Hongchen Sun Shimei Jiang PII: DOI: Reference:
S0925-4005(15)00737-6 http://dx.doi.org/doi:10.1016/j.snb.2015.06.004 SNB 18549
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
26-3-2015 23-5-2015 1-6-2015
Please cite this article as: C. Chen, G. Men, W. Bu, C. Liang, H. Sun, S. Jiang, A Colorimetric and Fluorescent Probe for Multiple Transition Metal Ions (Cu2+ , Zn2+ and Ni2+ ): Fast Response and High Selectivity, Sensors and Actuators B: Chemical (2015), http://dx.doi.org/10.1016/j.snb.2015.06.004 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.
Graphical Abstract (for review)
A Colorimetric and Fluorescent Probe for Multiple Transition Metal Ions (Cu2+, Zn2+ and Ni2+): Fast
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Response and High Selectivity Chunrong Chena, Guangwen Mena, Wenhuan Bub, Chunshuang Lianga, Hongchen Sunb,
an
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Shimei Jiang*a
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ed
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A multi-metal colorimetric and fluorescent sensor L, which could act as a fluorescent “turn on” sensor for Zn2+ and a colorimetric sensor for Cu2+, Zn2+ and Ni2+ in aqueous solution was developed.
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A Colorimetric and Fluorescent Probe for Multiple Transition Metal Ions (Cu2+, Zn2+ and Ni2+): Fast Response and High Selectivity
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Chunrong Chena, Guangwen Mena, Wenhuan Bub, Chunshuang Lianga, Hongchen
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Sunb, Shimei Jiang*a
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State Key Laboratory of Supramolecular Structure and Materials, Jilin University,
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2699 Qianjin Avenue, Changchun 130012, P. R. China.
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*Corresponding author: Shimei Jiang E-mail: [email protected]
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Tel: +86-431-85168474
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Fax: +86-431-85193421
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Abstract: A multi-metal colorimetric and fluorescent sensor based on Schiff base bearing an “O-N-N”-coordination site was developed. This newly designed sensor is a highly sensitive and selective “turn-on” fluorescent chemosensor towards Zn2+ which is less affected by physiologically relevant metal ions, especially Cd2+. The cell
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imaging experiment further confirmed that the sensor could be used for monitoring trace Zn2+ in living cells. On the other hand, evident color changes can be observed
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from colorless to yellow, orange and purple upon the sensor selective binding with
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Cu2+, Zn2+ and Ni2+, respectively. The corresponding color changes can be directly discriminated by “naked eye”. Significant response from spectral shift provided
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distinctly different profiles for each of the three metal ions. Overall, the sensor could simultaneously detect and differentiate three transition metal ions through fluorogenic
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(Zn2+) and chromogenic (Cu2+, Zn2+ and Ni2+) methods in real time and the detection
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limit was as low as 10-8 M in aqueous medium.
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Keywords: multi-metal sensor; Schiff base; cell imaging
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1. Introduction
Selective recognition and sensing of transition metal ions attract increasing studies
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due to their significant importance in chemical, biological, and environmental processes.1-3 Various transition metal chemosensors have been reported, especially the
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sensors based on the changes of fluorescence and color. These optical sensors were
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particularly attractive on account of their highly sensitive and selective, quick,
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inexpensive, easy to fabricate, and non-destructive properties.4-6 However, most
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chemosensors have been designed and developed to pursue high selectivity to only one metal ion, few of them are “one to more” type.7-10 Therefore, it is a real challenge
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to develop a chemosensor for simultaneously detecting and differentiating multi-metal
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ions. In addition, the detection of multiple targets with a single receptor would be more efficient, which can also lower down the cost compared to the one-to-one
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analysis method.11-17
Cu2+, Zn2+ and Ni2+ are essential trace elements in human body. The deficiency or excess of them can induce some serious diseases. Detection of them has long been realized as an important goal with a view to their important biological and environmental functions.18-22 On the periodic table, the three transition metal ions Cu2+, Zn2+ and Ni2+ line in the same period and next to each other. The high proximity 3
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of their values of ionic radius and charge density as well as similar coordinating ability of the aforesaid ions, make it very hard to distinguish their photophysical properties.20 Lots of chemosensors for each of them have been reported, but few have
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been developed for Cu2+, Zn2+ and Ni2+ simultaneously so far.23-25 Furthermore, most
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developed chemosensors were limited in organic solution and become invalid as soon
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as contacting with water.26-28 It remains a challenge to design a chemosensor that not
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only can detect but also differentiate Cu2+, Zn2+ and Ni2+, especially in aqueous
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solution.
Schiff bases are one kind of well known metal sensors due to their simple synthetic
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procedures, wide range of modifications and easy coordination with metal ions. 29-30
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In addition, transition metal complexes derived from Schiff base ligands have been widely studied due to their special biological activities, such as antiviral, anticancer, antibacterial, models for metalloenzyme active sites.31 Our group have reported a lot
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of optical chemosensors based on Schiff-base derivatives for various analytes.32 Most of the free Schiff base ligands were weakly fluorescent due to a photoinduced electron transfer (PET) process as well as a C=N isomerization.33 Binding with metal ions inhibits the isomerization of C=N, and enhances the fluorescence intensity through the CHEF (chelation-enhanced fluorescence) mechanism.33 On the other hand, the spectral changes and shifts from UV absorption provided different profiles for each of 4
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the transition metal ions. Therefore, Schiff bases have been widely used as metal ioncoordinated optical chemosensors, including fluorescent and colorimetric ones.
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Herein, we presented a novel chemosensor (L) obtained by simplely integrating salicylaldehyde and aminoquinoline groups. The sensor L has an “O-N-N”-
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coordination site (an oxygen atom of salicylaldehyde and two nitrogen atoms of
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aminoquinoline) (Scheme 1), which might act as a good combining center for
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detecting transition metal ions. Indeed, sensor L could act as a fluorescent “turn on”
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sensor for Zn2+, and a colorimetric sensor for Cu2+, Zn2+ and Ni2+. Most important, sensor L is an effective chemosensor for both detection and differentiation of Cu2+,
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Zn2+ and Ni2+. It is further confirmed that sensor L has potential practical applications,
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such as bio-imaging, through mapping of Zn2+ levels in cells.
2. Experimental section
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2.1 General information
All the reagents for synthesis and spectra were purchased from commercial suppliers (analytical grade) and used without further purification. Solutions of metal ions were prepared from the corresponding chloride. 1H NMR (TMS) and
13
C NMR spectras
were recorded on a Bruker UltraShield 500 MHz spectrometer and chemical shifts are recorded in ppm. Mass spectra were recorded on a Thermo Scientific ITQ 1100™GC/MSn and a Q Trap MS (Applied Biosystems/MDS Sciex, Concord, ON, 5
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Canada) which was equipped with an electrospray ionisation (ESI) source. The UVvis absorption spectra were taken on a Shimadzu 3100 UV-VIS-NIR recording spectrophotometer using a 5 nm slit width. The fluorescence spectra were recorded with a Shimadzu RF-5301PC spectrofluorophotometer. Elemental analyses were
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carried out with a vario MICRO cube elementar. Unless otherwise mentioned, all the
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measurements were performed at room temperature and repeated at least once.
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2.2 Synthesis of sensor L
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The Schiff base compound, 2, 4-di-tert-butyl-6-((quinolin-8-ylimino) methyl) phenol (L), was prepared according to the literature method. 3, 5-di-tert-butylsalicylaldehyde
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(0.234 g, 1 mmol) and 8-aminoquinoline (0.144 g, 1 mmol) were added into the
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solution of ethanol (30 mL). The resulting mixture was refluxed for 6 h and then cooled to room temperature. The orange precipitate was filtered and recrystallized,
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and dried in air to obtain the desired compound (0.3 g, 83%) 1H NMR (500 MHz, DMSO) δ 14.58 (s, 1H), 9.11 (s, 1H), 9.02 (dd, J = 4.1, 1.6 Hz, 1H), 8.45 (dd, J = 8.3,
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1.6 Hz, 1H), 7.94 (d, J = 7.4 Hz, 1H), 7.77 (d, J = 6.4 Hz, 1H), 7.70 (t, J = 7.8 Hz, 1H), 7.64 (dd, J = 8.3, 4.1 Hz, 1H), 7.53 (d, J = 2.4 Hz, 1H), 7.44 (d, J = 2.3 Hz, 1H). 13
C NMR (500 MHz, CDCl3) δ 166.35, 158.65, 150.00, 146.17, 142.07, 140.06,
137.03, 135.88, 131.44, 129.13, 128.04, 126.97, 126.47, 125.58, 121.41, 119.44, 35.34, 35.16, 34.53, 34.19, 31.51, 31.33, 29.53, 29.29. ESI-MS m/z: calculated for C24H28N2O (M + H+) : 361.2, found 361.3. Elemental analysis calculated for 6
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C24H28N2O: C, 79.96; H, 7.83; N, 7.77. Found: C, 81.12; H, 7.89; N, 7.84.
2.3 Spectral measurements
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Stock solutions of the metal ions (2 mM) were prepared in deionized water. A stock solution of L (2 mM) was prepared in ethanol. The solution of L was then diluted to
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50 μM with a H2O-ethanol (80 : 20 v/v) mixed solution. In titration experiments, each
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time, 2 mL solution of L (50 μM) was filled in a quartz optical cell of 1 cm optical
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path length, and the metal stock solution was added to the quartz optical cell gradually
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using a micro-pipette. Spectral data were recorded at 1 min after the addition. In selectivity experiments, the test samples were prepared by placing appropriate
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amounts of metal ion stock into 2 mL solution of L (50 μM). For fluorescence
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measurements, excitation was provided at 468 nm, and emission was collected from 480 to 750 nm
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2.4 Compete with other metal ions.
L (7.2 mg, 0.02 mmol) was dissolved in ethanol (10 mL) and 50 μL of this solution was diluted with 1.95 mL of H2O-ethanol (80 : 20 v/v) mixed solution to make the final concentration of 50 μM. MCl2 ( (M = Fe2+, Ni2+, Cu2+, Cd2+, Na+, K+, Li+, Mg2+, Ca2+, Al3+, Pb2+, Hg2+, Sr2+, Mn2+, Cu+ and Ba2+, 0.02 mmol) was dissolved in water (10 mL). 7
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For Zn2+ ion, 25 μL of each metal solution was taken and added to 2 mL of the solution of receptor L (50 μM) to give 0.5 equiv. of metal ions. Then, 25 μL of Zn2+ solution was added into the mixed solution of each metal ions and L to make 1
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equivalent. After mixing for a few seconds, fluorescence spectra were taken at room
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temperature.
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For Ni2+ ion, 25 μL of each metal solution was taken and added to 2 mL of the solution of receptor L (50 μM) to give 0.5 equiv. of metal ions. Then, 25 μL of Ni2+
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solution was added into the mixed solution of each metal ions and L to make 1 equivalent. After mixing them, UV-vis spectra were obtained at room temperature.
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For Cu2+ ion, 25 μL of each metal solution (20 mM) was taken and added to 2 mL of the solution of receptor L (50 μM) to give 0.5 equiv. of metal ions. Then, 25 μL of
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Cu2+ solution was added to the mixed solution of each metal ions and L to make 1
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equivalent. After mixing, UV-vis spectra were taken at room temperature.
2.5 Job plot measurements.
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L (7.2 mg, 0.02 mmol) was dissolved in ethanol (10 mL). 50, 45, 40, 35, 33, 30, 25, 20, 15, 10, 5 and 0 μL of the L solution were taken and transferred to vials. Zn2+, Cu2+, and Ni2+ (0.02 mmol) were dissolved in water (10 mL). 0, 5, 10, 15, 17, 20, 25, 30, 35, 40, 45 and 50 μL of the metal solution were added to each vial with L solution. Each vial was diluted with H2O-ethanol (80 : 20 v/v) mixed solution to make a total volume of 2 mL. After shaking, fluorescence or absorbance spectra were taken at room 8
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temperature.
2.6 Methods for cell culture HeLa human cervical carcinoma cells were cultured in Dulbecco’s Modified Eagle’s
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Medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco), and 1% penicillin-streptomycin solution (Hyclone). One day before imaging, the cells
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were platted on glass bottom cell culture dish. For labeling, the growth medium was
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removed. The cells were treated and incubated with 50 μL of L solved in ethanolHEPES (20 : 80 v/v) mixed solution at 37 °C under 5% CO2 for 30 min. Then, the
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cells were incubated for another 30 min under 50 μL of fresh medium that contained
3. Results and discussion
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three times with HEPES buffer.
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either 50 μM or 0 mM ZnCl2 mentioned above. Prior to imaging, cells were rinsed
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3.1 Design and synthesis of sensor L
Compound L was synthesized from 3,5-di-tert-butylsalicylaldehyde and 8-
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aminoquinoline by a simple aldehyde-amine condensation reaction in boiling ethanol (Scheme 1) using the procedure in the literature34 and the structure was confirmed by 1
H NMR, 13C NMR and mass spectra (Fig. S1-S3, ESI†). This newly designed sensor
L was an “O-N-N” type ligand containing three coordination sites which would increase the binding efficiency with metal ions.35 On the other hand, the typical electron pushing substituent tertiary butyl was introduced to improve the selectivity to 9
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metal ions.36 3.2 Response to Zn2+ The fluorescence response behavior of L was examined upon treatment with chloride
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salt of various metal ions in H2O-EtOH (8 : 2, v/v). Only Zn2+ caused a pronounced
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fluorescence enhancement at 584 nm, whereas the addition of 4 equiv. of other metal
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ions, such as Ba2+, K+, Na+, Li+, Al3+, Mg2+, Ca2+, Pb2+, Sr2+, Cd2+, Hg2+, Fe2+, Cu2+, Mn2+, Cu+ and Ni2+, caused no fluorescence enhancement (Fig. 1a). The sensor L
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solution can appear bright orange fluorescence color upon the addition of Zn2+ under
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a commercially available UV lamp at low concentration, the color change can be
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clearly observed by the naked eyes. L was supposed to be Zn2+-selective “turn-on” type of fluorescent chemosensor worked in aqueous solution. The selectivity of L
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towards various common metal ions was plotted as a bar graph in Fig. 1b. L is highly efficient in detecting Zn2+ over other metal ions commonly existing in physiological
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and environmental samples. Especially, L was not affected by Cd2+ which usually induced a comparable fluorescence response to that of Zn2+. The sensitivity of L was studied by fluorescence response towards various concentrations of Zn2+. Fig. 2 showed the changes in fluorescence spectra of L upon addition of Zn2+. While sensor L alone displayed negligible fluorescence emission, Zn2+ caused a prominent fluorescence enhancement at the wavelength of 584 nm and 10
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the intensity of the fluorescence peak had a 42-fold enhancement. The emission spectrum of L is quite sensitive to the presence of Zn2+. The emission intensity of L gradually enhanced with increasing the concentration of Zn2+, and was saturated at 0.5
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equiv. of Zn2+. The emission intensity at 584 nm was linearly proportional to the Zn2+
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concentration and the detection limit of L as a fluorescence chemosensor for Zn2+ was
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found to be as low as 1.5×10-8 M using the equation DL = 3σ/K (Fig. S4, ESI†). In the free status of Schiff base ligand, the nitrogen lone pair electrons at α position of the
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fluorophore are induced to the π system of the fluorophores, resulting in the
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quenching of their fluorescence. However, in the complex, the donation of the C=N
CHEF mechanism.33
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nitrogen lone pair to the metal center enhances the fluorescence intensity through the
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The binding properties of L with Zn2+ were further studied by UV-vis titration experiments (Fig. 3). Sensor L showed a strong absorbance band at 350 nm in H2O-
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EtOH (8 : 2, v/v). Upon gradual addition of Zn2+ the absorbance at 350 nm decreased and a new absorption band at 488 nm gradually increased and reached the maximum at 0.5 equiv. of Zn2+. Subsequently, the further addition of Zn2+ (>0.5 equiv.) did not change the absorbance spectrum. It indicates a clear transformation of the free ligand to its metal bound state. These observations support the formation of zinc complex. In order to verify the binding mode and the stoichiometry for L-Zn2+ complex, 11
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Job’s plot by using fluorescence data was carried out, the analytical result confirmed a 2 : 1 stoichiometry for the L-Zn2+ complex (Fig. S5, ESI†), which was highly consistent with the fluorescence and UV titration experiment results. More direct
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evidence was provided by the ESI mass spectra (Fig. S6, ESI†) of L upon the addition
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of Zn2+. In the presence of 0.5 equiv. of Zn2+ , the formation of a 2 : 1 complex
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between L and Zn2+ was confirmed by the emergence of the peak at m/z = 783.3 under positive ion mode. Furthermore, 1H NMR titrations verified the binding mode
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of L with Zn2+ (Fig. 4). Upon addition of Zn2+ to the receptor L, the proton on
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hydroxy Ha at 14.58 ppm disappeared completely. The proton on imine group Hb
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shifted to downfield from 9.10 ppm to 9.20 ppm, the other proton on the quinoline moiety Hc up-field shifted from 9.00 ppm to 8.80 ppm. In addition, all the aromatic
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protons displayed clear chemical shifts, indicating the participation of O atom and N atoms in bonding with the metal ions in the complex, supporting the assumption that
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L was an “O-N-N” type ligand. Based on the fluorescence and UV–vis titration experiments, ESI mass, 1H NMR spectra and Job’s plot analysis, the proposed structure of L-Zn2+ was shown in Scheme 2. To check the practical applicability of L as a Zn2+-selective fluorescence sensor, the anti-interference experiment with metal ions were carried out. (Fig. S7, ESI†). It was found that most of the metal ions did not interfere with the detection of Zn2+ by 12
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L, and only the common disruptor Fe2+, Cu2+, and Ni2+ which are paramagnetic metal ions lead to fluorescence quenching.37 Nevertheless, L had sufficient “turn-on” ratios for the detection of Zn2+ in the presence of biological abundant metal ions, like K+,
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Na+, Mg2+, Ca2+. These results mean that L had the potential to be a good sensor for
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Zn2+over other coexisting metal ions in cells.
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3.3 pH effect
To verify sensor L potential usage in biology, the pH effect on the fluorescence
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intensity is investigated by plotting the fluorescence intensity of L and L-Zn2+ vs. pH
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values, as shown in Fig. 5. An intense and stable fluorescence of L-Zn2+ in the pH range of 4.0-12.0 warrants its application under physiological conditions. Meanwhile,
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The fluorescence of L-Zn2+ was quenched under pH > 12 or pH< 4. When the pH is
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below 4, protonation of L inhibits it to coordinate with Zn2+ and the sensor L tended to be hydrolyzed at the low pH. If the pH is over 12, competition of OH- with L for
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binding to Zn2+ exists through the formation of Zn(OH)− or Zn(OH)2.38 Therefore, sensor L could be used in aqueous media of a broad pH range for Zn2+ detection. 3.4 Cell imaging
Since the sensor L exhibits excellent sensing properties for Zn2+ in vitro, subsequent experiments were conducted to test whether L can detect Zn2+ in live cells by fluorescence. HeLa cells were incubated with 50 μM L for 30 min at 37 °C, after
13
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three times washing with HEPES buffer, the cells were incubated with ZnCl2 (25 μM) for another 30 min. The results of the bright-field measurements (Fig. 6A and 6C) suggested that the cells were viable throughout the imaging experiments upon
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treatment with L and Zn2+, respectively. The HeLa cells that were cultured with L did
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not exhibit fluorescence, while those cells cultured with both L and Zn2+ exhibited
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strong intracellular fluorescence (Fig. 6B and 6D). These results suggested that L was
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membrane permeable and could respond to Zn2+ in live cells. Therefore, L can be supplied as a useful sensor for studying the distribution and physiological activity of
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3.5 Optical response to Cu2+ and Ni2+
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Zn2+ in live cells.
The chromogenic sensing ability of L was examined with various metal ions, such
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as Ba2+, K+, Na+, Li+, Al3+, Mg2+, Ca2+, Pb2+, Sr2+, Cd2+, Hg2+, Fe2+, Zn2+, Ni2+, Cu2+, Mn2+ and Cu+ in aqueous solution. As shown in Fig. 7, the sensor L showed evident
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color changes upon selective binding with Cu2+, Zn2+ and Ni2+. However, other metal ions did not cause any noticeable color and spectral changes under identical conditions. The color of sensor L changed from colorless to yellow, orange and purple upon binding with Cu2+, Zn2+ and Ni2+, respectively. Significant response from spectral shift provided distinctly different profiles for each of the three metal ions. The absorption at 488 nm caused by Zn2+ had been discussed above. The new 14
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absorption peak at 470 nm caused by Cu2+ was responsible for the yellow color of the solution. Ni2+ caused a broad absorption peak at 525 nm. These new peaks might be attributed to a metal-to-ligand-charge-transfer (MLCT) which was responsible for the
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yellow, orange and purple colors of the solutions.39 Metal Complex formation is on
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account of the coordination of metal ions with the lone-pair electrons on the nitrogen
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and oxygen atoms in aromatic cores. These atoms are very well known centers to
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coordinate with different transition metal ions. After binding with metal ions, the charge density of the aromatic cores changed and transferred thoroughly which will
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generate new absorption band in the UV-Vis spectra. Obviously, bind with metal ions
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with different charge density (Cu2+, Zn2+ and Ni2+) will cause different absorption peak in UV-Vis spectra. Therefore, L was supposed to be an efficient sensor to
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identify these three metal ions by “naked eyes”, respectively. The binding properties of L with Cu2+ were further studied by UV-Vis titration
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experiments. Upon addition of Cu2+ ions to the solution of L, the absorption band at 350 nm gradually decreased. At the same time a new absorption band at 470 nm significantly increased and reached maxima at 0.5 equiv. of Cu2+ (Fig. 8) which indicating a 2 : 1 ratio between L and Cu2+. Two clear isosbestic points were observed at 370 nm and 405 nm, implying the conversion of the free ligand L to the L-Cu2+ complex. Furthermore, the new absorption band in the visible region might be 15
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responsible to the color change from colorless to yellow. Job’s plot (Fig. S8, ESI†) analysis and ESI mass spectra (Fig. S9, ESI†) further confirmed the 2 : 1 stoichiometry for the L-Cu2+ complex (Scheme 2). In the MS spectra, the formation
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of a 2 : 1 complex between L and Cu2+ was confirmed by the emergence of the peak
concentration (linearly dependent coefficient: R2 =
t
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Cu2+
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at m/z = 782.6. The absorption intensity at 470 nm was linearly proportional to the
indicated that sensor L could be potentially used for quantitative detection of Cu2+
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concentration. The detection limit for Cu2+ was found to be as low as 1.76 × 10−7 M
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(Fig. S10, ESI†).
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The selectivity for Cu2+ by L was examined in the presence of 4 equiv. of other metal ions such as Ba2+, K+, Na+, Li+, Al3+, Mg2+, Ca2+, Pb2+, Sr2+, Cd2+, Hg2+, Fe2+,
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Zn2+, Ni2+, Mn2+ and Cu+. The absorption spectra of L remained almost unchanged (Fig. 9), indicating that none of the other metal ions interfered with the detection of
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Cu2+. The high selectivity may be due to its specific binding affinity of L toward Cu2+ over other metal ions. This result strongly indicated that receptor L is an excellent chemosensor for detection of Cu2+. The UV–visible titration experiment of L in the presence of Ni2+ was displayed in Fig. 10. The solution of L was colorless and had nearly no absorption at 525 nm. Upon addition of Ni2+, the solution changed from colorless to deep purple with a new 16
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absorption peak appearing at 525 nm. The significant spectra and color changes indicated that the free ligand had captured Ni2+. The absorbance gradually increased with the increasing of Ni2+ and was saturated at 0.5 equiv. of Ni2+, which mean the
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binding mode was the same as L-Zn2+. In addition, Job’s plot analysis (Fig.S11, ESI†)
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as well as ESI mass spectra (Fig. S12, ESI†) confirmed the 2: 1 stoichiometry for L-
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Ni2+ complex (Scheme 2). In the MS spectra, the formation of a 2 : 1 complex between L and Ni2+ was confirmed by the emergence of the peak at m/z = 777.5. The
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linear response of L to Ni2+ based on the absorption spectra had also been exploited.
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The detect limit was calculated to be 2.5 ×10-7 M (Fig. S13, ESI†). The results
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showed that L was a sensitive naked eyes indicator for Ni2+. In order to further evaluate the selectivity of L for Ni2+ sensing, competition
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experiments were carried out with other metal ions. The response of L to Ni2+ in the presence of 4 equiv. of various ions including Ba2+, K+, Na+, Li+, Al3+, Mg2+, Ca2+,
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Pb2+, Sr2+, Cd2+, Hg2+, Fe2+, Zn2+, Cu2+, Mn2+ and Cu+ were shown in Fig. 11. Most competing metal ions show no interference with Ni2+ detection, except for Cu2+ and Fe2+. Therefore, L was an excellent naked eyes sensor for Ni2+ in aqueous medium. According to the result of competition experiments for Ni2+ detection by L, most heavy metal ions didn’t perturbed the recognition of Ni2+ (Fig. 11). However, Cu2+ will cause fatal interference. Namely, when both Cu2+ and Ni2+ were in the sample, 17
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the sensor would give priority to Cu2+ rather than Ni2+. As shown in Scheme 3, in the presence of both Cu2+ and Ni2+, the color of the sensor L was changed from colorless to yellow which was the color of L-Cu2+ complex. After adding cysteine, the color
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changed from yellow to the color of L-Ni2+ purple.
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Cysteine with sulfydryl was the typical chelating agent for Cu2+, it was selected as
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the mask for Cu2+ due to its good performance in previous work. 40 In the presence of cysteine, the absorption band at 470 nm caused by Cu2+ was completely disappeared,
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and the color of L-Cu2+ was totally faded (Fig. 12). When both Cu2+ and Ni2+ were in
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the sample, the absorption band was the same with the complex of L-Cu2+. With the
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addition of cysteine, the UV spectra changed fatally, the absorption peak at 470 nm was fade away and the new absorption band at 525 nm for L-Ni2+ rose up. Thus, the
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color of the solution was controlled by the competitive coordination of Cu2+ and Ni2+ with sensor L in the presence or absence of cysteine. Therefore, the existence of
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cysteine was favorable for the selective detection of Ni2+ in the presence of Cu2+.
4. Conclusions
In summary, we have developed a highly effective metal sensor L, based on a Schiff base bearing an “O-N-N”-coordination site, for simultaneous detection of three transition metal ions (Zn2+, Ni2+ and Cu2+) through different signaling channels. This sensor showed a fluorescent “turn on” response to Zn2+ over other metal ions. Though 18
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the common disruptor Fe2+, Cu2+ and Ni2+ lead to fluorescence quenching, these free heavy metal ions are present at very low concentrations (nM) in cells and would not possibly interfere with the detection of Zn2+ in cell.41 Meanwhile, L was a
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colorimetric sensor to Cu2+, Zn2+ and Ni2+, respectively. When both of Cu2+ and Ni2+
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existed in the same sample, L preferred to bind with Cu2+ rather than Ni2+. In the
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presence of Cysteine, the sensor could be made selective towards Ni2+ over Cu2+. Cell imaging of confocal fluorescence microscopy further demonstrated that L can be used
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for mapping Zn2+ levels in living cells. The study further illustrates a novel strategy to
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differentiate the three transition metal ions (Cu2+, Zn2+ and Ni2+) by “naked eyes”.
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Thus, these results are significant and interesting for a new generation of metal recognition systems that can detect multiple analysts with one single small molecule
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without any complicated designs.
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Acknowledgment
This work was supported by the National Basic Research Program of China
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(2012CB933800) and the National Natural Science Foundation of China (21374036). Reference
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[1]. H. N. Kim, W. X. Ren, J. S. Kim and J. Yoon, Chem. Soc. Rev. 41 (2012)
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3210–3244.
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[2]. J. F. Zhang, Y. Zhou, J. Yoon, J. S. Kim, Chem. Soc. Rev. 40 (2011) 3416.
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[3]. G. Aragay, J. Pons, A. Merkoci, Chem. Rev. 111 (2011) 3433-3458. [4]. J. S. Wu, W. M. Liu, J. C. Ge, H. Y. Zhang, P. F. Wang, Chem. Soc. Rev. 40
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Author Biographies Chunrong Chen received her BSc degree in chemistry from Sichuan University in 2013. Currently she is a graduate student of the College of Chemistry in Jilin
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University.
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Guangwen Men received his PhD degree in chemistry from Jilin University,
Bu received
his
BSc
degree
in
School
of
Stomatology
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Wenhuan
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Changchun, China, in 2014. Currently he is a post doctor at Jilin University.
ed
from Jilin University in 2014. Currently he is a graduate student of the School of
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Stomatology in Jilin University.
Chunshuang Liang received her BSc degree in chemistry from Jilin University in
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2012. Currently she is a graduate student of the College of Chemistry in Jilin University.
Hongchen Sun is a professor of School of Stomatology at Jilin University. He received his PhD degree from Norman Bethune University of Medical Sciences in 1998. His main research interests are hard tissue remodeling and regeneration, 25
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preparation and application of biomaterials for bone regeneration.
Shimei Jiang is a professor of Chemistry at Jilin University. She received her PhD
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responsive supramolecular systems and nano-materials.
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degree from Jilin University in 1998. Her main research interests are stimuli-
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Figure captions:
Scheme 1 Synthetic route of compound L. Fig. 1 Fluorescence spectra (λex = 468 nm) (a) and bar graph (b) of L (50 μM) before
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and after addition of metal ions of (200 μM) Zn2+, Ba2+, K+, Na+, Li+, Al3+, Mg2+, Ca2+, Pb2+, Sr2+, Cd2+, Hg2+, Fe2+, Cu2+, Mn2+, Cu+ and Ni2+ in H2O-EtOH (8 : 2, v/v).
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The photos of L and L+Zn2+ were taken under UV light.
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Fig. 2 Fluorescence spectra of L (50 μM, λex = 468 nm) upon the addition of
increasing amounts of Zn2+ ions (0, 0.5, 1, 1.5, 3, 4, 5, 10, 15, 20, 25, 30, and 40 μM)
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in H2O-EtOH (8 : 2, v/v). The arrow indicates the change in the emission intensity with the increased Zn2+ ions. Inset: intensity at 584 nm versus the concentration of
M
Zn2+ added.
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Fig. 3 UV-vis spectra of L (50 μM) upon the addition of zinc ions (0, 0.5, 1, 1.5, 3, 4, 5, 6, 8, 10, 25, and 50μM) in H2O-EtOH (8 : 2, v/v). The arrow indicates the change
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in the absorbance with the increased Zn2+ ions. Inset: absorbance at 500 nm versus the concentration of Zn2+ added.
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Fig. 4 1H NMR of L and L-Zn2+ in DMSO-d6 : a) L-Zn2+, b) only L. Scheme 2 Proposed structures of L-M2+ complexes (M = Zn, Cu and Ni). Fig. 5 Fluorescence responses of L and L-Zn2+ at various ranges of pH in aqueous solution (H2O: EtOH = 8 : 2, v/v). λex= 468 nm. Fig. 6 Fluorescence images of Hela cell incubated with L and Zn2+. Cells were incubated with 50 μM L for 30 min (A and B) and then further with 25 μM ZnCl2 for 30 min (C and D). The left images (A, C) were observed with the light microscope 27
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and the right images (B, D) were taken with a fluorescence microscope. The scale bar is 50 μm. Fig. 7 (a) Absorption spectra of L (50 μM) in the presence of 200 μM various metal ions in aqueous solution (H2O: EtOH = 8 : 2, v/v). (b) The color of L (50 μM) upon
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addition of various metal ions .
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Fig. 8 UV-vis spectra of L (50 μM) upon the addition of Cu2+ (0, 1, 4, 6, 7, 10, 12, 14, 16, 18, 20, 22, 25, and 30 μM) in aqueous solution (H2O: EtOH = 8 : 2, v/v). The
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arrow indicates the change in the absorbance with the increased Cu2+ ions. Inset:
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absorbance at 470 nm versus the concentration of Cu2+ added.
Fig. 9 Effect of competitive metal ions (25 μM) on the interaction between sensor L
M
(50 μM) and Cu2+ ion (25 μM) in aqueous solution (H2O: EtOH = 8 : 2, v/v).
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Fig. 10 UV–vis spectra of L (50 μM) upon the addition of Ni2+ (0, 1, 2, 3, 4, 5, 6, 8, 10, 15, 18, 20, and 25 μM) in aqueous solution (H2O: EtOH = 8 : 2, v/v). The arrow
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indicates the change in the absorbance with the increased Ni2+ ions. Inset: absorbance at 525 nm versus the concentration of Ni2+ added. Fig. 11 Effect of competitive metal ions (25 μM) on the interaction between sensor L
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(50 μM) and Ni2+ ion (25 μM) in aqueous solution (H2O: EtOH = 8 : 2, v/v). Scheme 3 Competitive coordination pattern of Cu2+ and Ni2+ with L. Fig. 12 UV–vis spectra of competitive coordination of Cu2+and Ni2+ with L in the presence and absence of cysteine in aqueous solution (H2O: EtOH = 8 : 2, v/v).
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Scheme 1 Synthetic route of compound L.
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Fig. 1 Fluorescence spectra (λex = 468 nm) (a) and bar graph (b) of L (50 μM) before and after addition of metal ions of (200 μM) Zn2+, Ba2+, K+, Na+, Li+, Al3+, Mg2+, Ca2+, Pb2+, Sr2+, Cd2+, Hg2+, Fe2+, Cu2+, Mn2+, Cu+ and Ni2+ in H2O-EtOH (8 : 2, v/v).
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The photos of L and L+Zn2+ were taken under UV light.
30
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Fig. 2 Fluorescence spectra of L (50 μM, λex = 468 nm) upon the addition of increasing amounts of Zn2+ ions (0, 0.5, 1, 1.5, 3, 4, 5, 10, 15, 20, 25, 30, and 40μM) in H2O-EtOH (8 : 2, v/v). The arrow indicates the change in the emission intensity
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with the increased Zn2+ ions. Inset: intensity at 584 nm versus the concentration of
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Zn2+ added.
31
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Fig. 3 UV-vis spectra of L (50 μM) upon the addition of zinc ions (0, 0.5, 1, 1.5, 3, 4, 5, 6, 8, 10, 25, and 50μM) in H2O-EtOH (8 : 2, v/v). The arrow indicates the change in the absorbance with the increased Zn2+ ions. Inset: absorbance at 500 nm versus the
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ed
M
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concentration of Zn2+ added.
32
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Fig. 4 1H NMR of L and L-Zn2+ in DMSO-d6 : a) L-Zn2+, b) only L.
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Scheme 2 Proposed structures of L-M2+ complexes (M = Zn, Cu and Ni).
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Fig. 5 Fluorescence responses of L and L-Zn2+ at various ranges of pH in aqueous
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solution (H2O: EtOH = 8 : 2, v/v). λex= 468 nm.
35
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Fig. 6 Fluorescence images of Hela cell incubated with L and Zn2+. Cells were incubated with 50 μM L for 30 min (A and B) and then further with 25 μM ZnCl2 for 30 min (C and D). The left images (A, C) were observed with the light microscope and the right images (B, D) were taken with a fluorescence microscope. The scale bar
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is 50 μm.
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Fig. 7 (a) Absorption spectra of L (50 μM) in the presence of 200 μM various metal ions in aqueous solution (H2O: EtOH = 8 : 2, v/v). (b) The color of L (50 μM) upon
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addition of various metal ions .
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Fig. 8 UV-vis spectra of L (50 μM) upon the addition of Cu2+ (0, 1, 4, 6, 7, 10, 12, 14, 16, 18, 20, 22, 25, and 30 μM) in aqueous solution (H2O: EtOH = 8 : 2, v/v). The arrow indicates the change in the absorbance with the increased Cu2+ ions. Inset:
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ed
M
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absorbance at 470 nm versus the concentration of Cu2+ added.
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Fig. 9 Effect of competitive metal ions (25 μM) on the interaction between sensor L
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ed
M
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(50 μM) and Cu2+ ion (25 μM) in aqueous solution (H2O: EtOH = 8 : 2, v/v).
39
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Fig. 10 UV–vis spectra of L (50 μM) upon the addition of Ni2+ (0, 1, 2, 3, 4, 5, 6, 8, 10, 15, 18, 20, and 25 μM) in aqueous solution (H2O: EtOH = 8 : 2, v/v). The arrow indicates the change in the absorbance with the increased Ni2+ ions. Inset: absorbance
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M
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at 525 nm versus the concentration of Ni2+ added.
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Fig. 11 Effect of competitive metal ions (25 μM) on the interaction between sensor L
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(50 μM) and Ni2+ ion (25 μM) in aqueous solution (H2O: EtOH = 8 : 2, v/v).
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Scheme 3 Competitive coordination pattern of Cu2+ and Ni2+ with L.
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Fig. 12 UV–vis spectra of competitive coordination of Cu2+and Ni2+ with L in the
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M
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presence and absence of cysteine in aqueous solution (H2O: EtOH = 8 : 2, v/v).
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S0925-4005(15)00737-6 http://dx.doi.org/doi:10.1016/j.snb.2015.06.004 SNB 18549
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
26-3-2015 23-5-2015 1-6-2015
Please cite this article as: C. Chen, G. Men, W. Bu, C. Liang, H. Sun, S. Jiang, A Colorimetric and Fluorescent Probe for Multiple Transition Metal Ions (Cu2+ , Zn2+ and Ni2+ ): Fast Response and High Selectivity, Sensors and Actuators B: Chemical (2015), http://dx.doi.org/10.1016/j.snb.2015.06.004 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.
Graphical Abstract (for review)
A Colorimetric and Fluorescent Probe for Multiple Transition Metal Ions (Cu2+, Zn2+ and Ni2+): Fast
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Response and High Selectivity Chunrong Chena, Guangwen Mena, Wenhuan Bub, Chunshuang Lianga, Hongchen Sunb,
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Shimei Jiang*a
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A multi-metal colorimetric and fluorescent sensor L, which could act as a fluorescent “turn on” sensor for Zn2+ and a colorimetric sensor for Cu2+, Zn2+ and Ni2+ in aqueous solution was developed.
Page 1 of 44
A Colorimetric and Fluorescent Probe for Multiple Transition Metal Ions (Cu2+, Zn2+ and Ni2+): Fast Response and High Selectivity
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Chunrong Chena, Guangwen Mena, Wenhuan Bub, Chunshuang Lianga, Hongchen
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Sunb, Shimei Jiang*a
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State Key Laboratory of Supramolecular Structure and Materials, Jilin University,
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2699 Qianjin Avenue, Changchun 130012, P. R. China.
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*Corresponding author: Shimei Jiang E-mail: [email protected]
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Tel: +86-431-85168474
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Fax: +86-431-85193421
1
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Abstract: A multi-metal colorimetric and fluorescent sensor based on Schiff base bearing an “O-N-N”-coordination site was developed. This newly designed sensor is a highly sensitive and selective “turn-on” fluorescent chemosensor towards Zn2+ which is less affected by physiologically relevant metal ions, especially Cd2+. The cell
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imaging experiment further confirmed that the sensor could be used for monitoring trace Zn2+ in living cells. On the other hand, evident color changes can be observed
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from colorless to yellow, orange and purple upon the sensor selective binding with
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Cu2+, Zn2+ and Ni2+, respectively. The corresponding color changes can be directly discriminated by “naked eye”. Significant response from spectral shift provided
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distinctly different profiles for each of the three metal ions. Overall, the sensor could simultaneously detect and differentiate three transition metal ions through fluorogenic
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(Zn2+) and chromogenic (Cu2+, Zn2+ and Ni2+) methods in real time and the detection
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limit was as low as 10-8 M in aqueous medium.
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Keywords: multi-metal sensor; Schiff base; cell imaging
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1. Introduction
Selective recognition and sensing of transition metal ions attract increasing studies
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due to their significant importance in chemical, biological, and environmental processes.1-3 Various transition metal chemosensors have been reported, especially the
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sensors based on the changes of fluorescence and color. These optical sensors were
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particularly attractive on account of their highly sensitive and selective, quick,
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inexpensive, easy to fabricate, and non-destructive properties.4-6 However, most
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chemosensors have been designed and developed to pursue high selectivity to only one metal ion, few of them are “one to more” type.7-10 Therefore, it is a real challenge
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to develop a chemosensor for simultaneously detecting and differentiating multi-metal
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ions. In addition, the detection of multiple targets with a single receptor would be more efficient, which can also lower down the cost compared to the one-to-one
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analysis method.11-17
Cu2+, Zn2+ and Ni2+ are essential trace elements in human body. The deficiency or excess of them can induce some serious diseases. Detection of them has long been realized as an important goal with a view to their important biological and environmental functions.18-22 On the periodic table, the three transition metal ions Cu2+, Zn2+ and Ni2+ line in the same period and next to each other. The high proximity 3
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of their values of ionic radius and charge density as well as similar coordinating ability of the aforesaid ions, make it very hard to distinguish their photophysical properties.20 Lots of chemosensors for each of them have been reported, but few have
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been developed for Cu2+, Zn2+ and Ni2+ simultaneously so far.23-25 Furthermore, most
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developed chemosensors were limited in organic solution and become invalid as soon
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as contacting with water.26-28 It remains a challenge to design a chemosensor that not
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only can detect but also differentiate Cu2+, Zn2+ and Ni2+, especially in aqueous
M
solution.
Schiff bases are one kind of well known metal sensors due to their simple synthetic
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procedures, wide range of modifications and easy coordination with metal ions. 29-30
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In addition, transition metal complexes derived from Schiff base ligands have been widely studied due to their special biological activities, such as antiviral, anticancer, antibacterial, models for metalloenzyme active sites.31 Our group have reported a lot
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of optical chemosensors based on Schiff-base derivatives for various analytes.32 Most of the free Schiff base ligands were weakly fluorescent due to a photoinduced electron transfer (PET) process as well as a C=N isomerization.33 Binding with metal ions inhibits the isomerization of C=N, and enhances the fluorescence intensity through the CHEF (chelation-enhanced fluorescence) mechanism.33 On the other hand, the spectral changes and shifts from UV absorption provided different profiles for each of 4
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the transition metal ions. Therefore, Schiff bases have been widely used as metal ioncoordinated optical chemosensors, including fluorescent and colorimetric ones.
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Herein, we presented a novel chemosensor (L) obtained by simplely integrating salicylaldehyde and aminoquinoline groups. The sensor L has an “O-N-N”-
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coordination site (an oxygen atom of salicylaldehyde and two nitrogen atoms of
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aminoquinoline) (Scheme 1), which might act as a good combining center for
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detecting transition metal ions. Indeed, sensor L could act as a fluorescent “turn on”
M
sensor for Zn2+, and a colorimetric sensor for Cu2+, Zn2+ and Ni2+. Most important, sensor L is an effective chemosensor for both detection and differentiation of Cu2+,
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Zn2+ and Ni2+. It is further confirmed that sensor L has potential practical applications,
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such as bio-imaging, through mapping of Zn2+ levels in cells.
2. Experimental section
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2.1 General information
All the reagents for synthesis and spectra were purchased from commercial suppliers (analytical grade) and used without further purification. Solutions of metal ions were prepared from the corresponding chloride. 1H NMR (TMS) and
13
C NMR spectras
were recorded on a Bruker UltraShield 500 MHz spectrometer and chemical shifts are recorded in ppm. Mass spectra were recorded on a Thermo Scientific ITQ 1100™GC/MSn and a Q Trap MS (Applied Biosystems/MDS Sciex, Concord, ON, 5
Page 6 of 44
Canada) which was equipped with an electrospray ionisation (ESI) source. The UVvis absorption spectra were taken on a Shimadzu 3100 UV-VIS-NIR recording spectrophotometer using a 5 nm slit width. The fluorescence spectra were recorded with a Shimadzu RF-5301PC spectrofluorophotometer. Elemental analyses were
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carried out with a vario MICRO cube elementar. Unless otherwise mentioned, all the
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measurements were performed at room temperature and repeated at least once.
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2.2 Synthesis of sensor L
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The Schiff base compound, 2, 4-di-tert-butyl-6-((quinolin-8-ylimino) methyl) phenol (L), was prepared according to the literature method. 3, 5-di-tert-butylsalicylaldehyde
M
(0.234 g, 1 mmol) and 8-aminoquinoline (0.144 g, 1 mmol) were added into the
ed
solution of ethanol (30 mL). The resulting mixture was refluxed for 6 h and then cooled to room temperature. The orange precipitate was filtered and recrystallized,
ce pt
and dried in air to obtain the desired compound (0.3 g, 83%) 1H NMR (500 MHz, DMSO) δ 14.58 (s, 1H), 9.11 (s, 1H), 9.02 (dd, J = 4.1, 1.6 Hz, 1H), 8.45 (dd, J = 8.3,
Ac
1.6 Hz, 1H), 7.94 (d, J = 7.4 Hz, 1H), 7.77 (d, J = 6.4 Hz, 1H), 7.70 (t, J = 7.8 Hz, 1H), 7.64 (dd, J = 8.3, 4.1 Hz, 1H), 7.53 (d, J = 2.4 Hz, 1H), 7.44 (d, J = 2.3 Hz, 1H). 13
C NMR (500 MHz, CDCl3) δ 166.35, 158.65, 150.00, 146.17, 142.07, 140.06,
137.03, 135.88, 131.44, 129.13, 128.04, 126.97, 126.47, 125.58, 121.41, 119.44, 35.34, 35.16, 34.53, 34.19, 31.51, 31.33, 29.53, 29.29. ESI-MS m/z: calculated for C24H28N2O (M + H+) : 361.2, found 361.3. Elemental analysis calculated for 6
Page 7 of 44
C24H28N2O: C, 79.96; H, 7.83; N, 7.77. Found: C, 81.12; H, 7.89; N, 7.84.
2.3 Spectral measurements
ip t
Stock solutions of the metal ions (2 mM) were prepared in deionized water. A stock solution of L (2 mM) was prepared in ethanol. The solution of L was then diluted to
cr
50 μM with a H2O-ethanol (80 : 20 v/v) mixed solution. In titration experiments, each
us
time, 2 mL solution of L (50 μM) was filled in a quartz optical cell of 1 cm optical
an
path length, and the metal stock solution was added to the quartz optical cell gradually
M
using a micro-pipette. Spectral data were recorded at 1 min after the addition. In selectivity experiments, the test samples were prepared by placing appropriate
ed
amounts of metal ion stock into 2 mL solution of L (50 μM). For fluorescence
ce pt
measurements, excitation was provided at 468 nm, and emission was collected from 480 to 750 nm
Ac
2.4 Compete with other metal ions.
L (7.2 mg, 0.02 mmol) was dissolved in ethanol (10 mL) and 50 μL of this solution was diluted with 1.95 mL of H2O-ethanol (80 : 20 v/v) mixed solution to make the final concentration of 50 μM. MCl2 ( (M = Fe2+, Ni2+, Cu2+, Cd2+, Na+, K+, Li+, Mg2+, Ca2+, Al3+, Pb2+, Hg2+, Sr2+, Mn2+, Cu+ and Ba2+, 0.02 mmol) was dissolved in water (10 mL). 7
Page 8 of 44
For Zn2+ ion, 25 μL of each metal solution was taken and added to 2 mL of the solution of receptor L (50 μM) to give 0.5 equiv. of metal ions. Then, 25 μL of Zn2+ solution was added into the mixed solution of each metal ions and L to make 1
ip t
equivalent. After mixing for a few seconds, fluorescence spectra were taken at room
cr
temperature.
us
For Ni2+ ion, 25 μL of each metal solution was taken and added to 2 mL of the solution of receptor L (50 μM) to give 0.5 equiv. of metal ions. Then, 25 μL of Ni2+
an
solution was added into the mixed solution of each metal ions and L to make 1 equivalent. After mixing them, UV-vis spectra were obtained at room temperature.
M
For Cu2+ ion, 25 μL of each metal solution (20 mM) was taken and added to 2 mL of the solution of receptor L (50 μM) to give 0.5 equiv. of metal ions. Then, 25 μL of
ed
Cu2+ solution was added to the mixed solution of each metal ions and L to make 1
ce pt
equivalent. After mixing, UV-vis spectra were taken at room temperature.
2.5 Job plot measurements.
Ac
L (7.2 mg, 0.02 mmol) was dissolved in ethanol (10 mL). 50, 45, 40, 35, 33, 30, 25, 20, 15, 10, 5 and 0 μL of the L solution were taken and transferred to vials. Zn2+, Cu2+, and Ni2+ (0.02 mmol) were dissolved in water (10 mL). 0, 5, 10, 15, 17, 20, 25, 30, 35, 40, 45 and 50 μL of the metal solution were added to each vial with L solution. Each vial was diluted with H2O-ethanol (80 : 20 v/v) mixed solution to make a total volume of 2 mL. After shaking, fluorescence or absorbance spectra were taken at room 8
Page 9 of 44
temperature.
2.6 Methods for cell culture HeLa human cervical carcinoma cells were cultured in Dulbecco’s Modified Eagle’s
ip t
Medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco), and 1% penicillin-streptomycin solution (Hyclone). One day before imaging, the cells
cr
were platted on glass bottom cell culture dish. For labeling, the growth medium was
us
removed. The cells were treated and incubated with 50 μL of L solved in ethanolHEPES (20 : 80 v/v) mixed solution at 37 °C under 5% CO2 for 30 min. Then, the
an
cells were incubated for another 30 min under 50 μL of fresh medium that contained
3. Results and discussion
ed
three times with HEPES buffer.
M
either 50 μM or 0 mM ZnCl2 mentioned above. Prior to imaging, cells were rinsed
ce pt
3.1 Design and synthesis of sensor L
Compound L was synthesized from 3,5-di-tert-butylsalicylaldehyde and 8-
Ac
aminoquinoline by a simple aldehyde-amine condensation reaction in boiling ethanol (Scheme 1) using the procedure in the literature34 and the structure was confirmed by 1
H NMR, 13C NMR and mass spectra (Fig. S1-S3, ESI†). This newly designed sensor
L was an “O-N-N” type ligand containing three coordination sites which would increase the binding efficiency with metal ions.35 On the other hand, the typical electron pushing substituent tertiary butyl was introduced to improve the selectivity to 9
Page 10 of 44
metal ions.36 3.2 Response to Zn2+ The fluorescence response behavior of L was examined upon treatment with chloride
ip t
salt of various metal ions in H2O-EtOH (8 : 2, v/v). Only Zn2+ caused a pronounced
cr
fluorescence enhancement at 584 nm, whereas the addition of 4 equiv. of other metal
us
ions, such as Ba2+, K+, Na+, Li+, Al3+, Mg2+, Ca2+, Pb2+, Sr2+, Cd2+, Hg2+, Fe2+, Cu2+, Mn2+, Cu+ and Ni2+, caused no fluorescence enhancement (Fig. 1a). The sensor L
an
solution can appear bright orange fluorescence color upon the addition of Zn2+ under
M
a commercially available UV lamp at low concentration, the color change can be
ed
clearly observed by the naked eyes. L was supposed to be Zn2+-selective “turn-on” type of fluorescent chemosensor worked in aqueous solution. The selectivity of L
ce pt
towards various common metal ions was plotted as a bar graph in Fig. 1b. L is highly efficient in detecting Zn2+ over other metal ions commonly existing in physiological
Ac
and environmental samples. Especially, L was not affected by Cd2+ which usually induced a comparable fluorescence response to that of Zn2+. The sensitivity of L was studied by fluorescence response towards various concentrations of Zn2+. Fig. 2 showed the changes in fluorescence spectra of L upon addition of Zn2+. While sensor L alone displayed negligible fluorescence emission, Zn2+ caused a prominent fluorescence enhancement at the wavelength of 584 nm and 10
Page 11 of 44
the intensity of the fluorescence peak had a 42-fold enhancement. The emission spectrum of L is quite sensitive to the presence of Zn2+. The emission intensity of L gradually enhanced with increasing the concentration of Zn2+, and was saturated at 0.5
ip t
equiv. of Zn2+. The emission intensity at 584 nm was linearly proportional to the Zn2+
cr
concentration and the detection limit of L as a fluorescence chemosensor for Zn2+ was
us
found to be as low as 1.5×10-8 M using the equation DL = 3σ/K (Fig. S4, ESI†). In the free status of Schiff base ligand, the nitrogen lone pair electrons at α position of the
an
fluorophore are induced to the π system of the fluorophores, resulting in the
M
quenching of their fluorescence. However, in the complex, the donation of the C=N
CHEF mechanism.33
ed
nitrogen lone pair to the metal center enhances the fluorescence intensity through the
ce pt
The binding properties of L with Zn2+ were further studied by UV-vis titration experiments (Fig. 3). Sensor L showed a strong absorbance band at 350 nm in H2O-
Ac
EtOH (8 : 2, v/v). Upon gradual addition of Zn2+ the absorbance at 350 nm decreased and a new absorption band at 488 nm gradually increased and reached the maximum at 0.5 equiv. of Zn2+. Subsequently, the further addition of Zn2+ (>0.5 equiv.) did not change the absorbance spectrum. It indicates a clear transformation of the free ligand to its metal bound state. These observations support the formation of zinc complex. In order to verify the binding mode and the stoichiometry for L-Zn2+ complex, 11
Page 12 of 44
Job’s plot by using fluorescence data was carried out, the analytical result confirmed a 2 : 1 stoichiometry for the L-Zn2+ complex (Fig. S5, ESI†), which was highly consistent with the fluorescence and UV titration experiment results. More direct
ip t
evidence was provided by the ESI mass spectra (Fig. S6, ESI†) of L upon the addition
cr
of Zn2+. In the presence of 0.5 equiv. of Zn2+ , the formation of a 2 : 1 complex
us
between L and Zn2+ was confirmed by the emergence of the peak at m/z = 783.3 under positive ion mode. Furthermore, 1H NMR titrations verified the binding mode
an
of L with Zn2+ (Fig. 4). Upon addition of Zn2+ to the receptor L, the proton on
M
hydroxy Ha at 14.58 ppm disappeared completely. The proton on imine group Hb
ed
shifted to downfield from 9.10 ppm to 9.20 ppm, the other proton on the quinoline moiety Hc up-field shifted from 9.00 ppm to 8.80 ppm. In addition, all the aromatic
ce pt
protons displayed clear chemical shifts, indicating the participation of O atom and N atoms in bonding with the metal ions in the complex, supporting the assumption that
Ac
L was an “O-N-N” type ligand. Based on the fluorescence and UV–vis titration experiments, ESI mass, 1H NMR spectra and Job’s plot analysis, the proposed structure of L-Zn2+ was shown in Scheme 2. To check the practical applicability of L as a Zn2+-selective fluorescence sensor, the anti-interference experiment with metal ions were carried out. (Fig. S7, ESI†). It was found that most of the metal ions did not interfere with the detection of Zn2+ by 12
Page 13 of 44
L, and only the common disruptor Fe2+, Cu2+, and Ni2+ which are paramagnetic metal ions lead to fluorescence quenching.37 Nevertheless, L had sufficient “turn-on” ratios for the detection of Zn2+ in the presence of biological abundant metal ions, like K+,
ip t
Na+, Mg2+, Ca2+. These results mean that L had the potential to be a good sensor for
cr
Zn2+over other coexisting metal ions in cells.
us
3.3 pH effect
To verify sensor L potential usage in biology, the pH effect on the fluorescence
an
intensity is investigated by plotting the fluorescence intensity of L and L-Zn2+ vs. pH
M
values, as shown in Fig. 5. An intense and stable fluorescence of L-Zn2+ in the pH range of 4.0-12.0 warrants its application under physiological conditions. Meanwhile,
ed
The fluorescence of L-Zn2+ was quenched under pH > 12 or pH< 4. When the pH is
ce pt
below 4, protonation of L inhibits it to coordinate with Zn2+ and the sensor L tended to be hydrolyzed at the low pH. If the pH is over 12, competition of OH- with L for
Ac
binding to Zn2+ exists through the formation of Zn(OH)− or Zn(OH)2.38 Therefore, sensor L could be used in aqueous media of a broad pH range for Zn2+ detection. 3.4 Cell imaging
Since the sensor L exhibits excellent sensing properties for Zn2+ in vitro, subsequent experiments were conducted to test whether L can detect Zn2+ in live cells by fluorescence. HeLa cells were incubated with 50 μM L for 30 min at 37 °C, after
13
Page 14 of 44
three times washing with HEPES buffer, the cells were incubated with ZnCl2 (25 μM) for another 30 min. The results of the bright-field measurements (Fig. 6A and 6C) suggested that the cells were viable throughout the imaging experiments upon
ip t
treatment with L and Zn2+, respectively. The HeLa cells that were cultured with L did
cr
not exhibit fluorescence, while those cells cultured with both L and Zn2+ exhibited
us
strong intracellular fluorescence (Fig. 6B and 6D). These results suggested that L was
an
membrane permeable and could respond to Zn2+ in live cells. Therefore, L can be supplied as a useful sensor for studying the distribution and physiological activity of
ed
3.5 Optical response to Cu2+ and Ni2+
M
Zn2+ in live cells.
The chromogenic sensing ability of L was examined with various metal ions, such
ce pt
as Ba2+, K+, Na+, Li+, Al3+, Mg2+, Ca2+, Pb2+, Sr2+, Cd2+, Hg2+, Fe2+, Zn2+, Ni2+, Cu2+, Mn2+ and Cu+ in aqueous solution. As shown in Fig. 7, the sensor L showed evident
Ac
color changes upon selective binding with Cu2+, Zn2+ and Ni2+. However, other metal ions did not cause any noticeable color and spectral changes under identical conditions. The color of sensor L changed from colorless to yellow, orange and purple upon binding with Cu2+, Zn2+ and Ni2+, respectively. Significant response from spectral shift provided distinctly different profiles for each of the three metal ions. The absorption at 488 nm caused by Zn2+ had been discussed above. The new 14
Page 15 of 44
absorption peak at 470 nm caused by Cu2+ was responsible for the yellow color of the solution. Ni2+ caused a broad absorption peak at 525 nm. These new peaks might be attributed to a metal-to-ligand-charge-transfer (MLCT) which was responsible for the
ip t
yellow, orange and purple colors of the solutions.39 Metal Complex formation is on
cr
account of the coordination of metal ions with the lone-pair electrons on the nitrogen
us
and oxygen atoms in aromatic cores. These atoms are very well known centers to
an
coordinate with different transition metal ions. After binding with metal ions, the charge density of the aromatic cores changed and transferred thoroughly which will
M
generate new absorption band in the UV-Vis spectra. Obviously, bind with metal ions
ed
with different charge density (Cu2+, Zn2+ and Ni2+) will cause different absorption peak in UV-Vis spectra. Therefore, L was supposed to be an efficient sensor to
ce pt
identify these three metal ions by “naked eyes”, respectively. The binding properties of L with Cu2+ were further studied by UV-Vis titration
Ac
experiments. Upon addition of Cu2+ ions to the solution of L, the absorption band at 350 nm gradually decreased. At the same time a new absorption band at 470 nm significantly increased and reached maxima at 0.5 equiv. of Cu2+ (Fig. 8) which indicating a 2 : 1 ratio between L and Cu2+. Two clear isosbestic points were observed at 370 nm and 405 nm, implying the conversion of the free ligand L to the L-Cu2+ complex. Furthermore, the new absorption band in the visible region might be 15
Page 16 of 44
responsible to the color change from colorless to yellow. Job’s plot (Fig. S8, ESI†) analysis and ESI mass spectra (Fig. S9, ESI†) further confirmed the 2 : 1 stoichiometry for the L-Cu2+ complex (Scheme 2). In the MS spectra, the formation
ip t
of a 2 : 1 complex between L and Cu2+ was confirmed by the emergence of the peak
concentration (linearly dependent coefficient: R2 =
t
us
Cu2+
cr
at m/z = 782.6. The absorption intensity at 470 nm was linearly proportional to the
indicated that sensor L could be potentially used for quantitative detection of Cu2+
an
concentration. The detection limit for Cu2+ was found to be as low as 1.76 × 10−7 M
M
(Fig. S10, ESI†).
ed
The selectivity for Cu2+ by L was examined in the presence of 4 equiv. of other metal ions such as Ba2+, K+, Na+, Li+, Al3+, Mg2+, Ca2+, Pb2+, Sr2+, Cd2+, Hg2+, Fe2+,
ce pt
Zn2+, Ni2+, Mn2+ and Cu+. The absorption spectra of L remained almost unchanged (Fig. 9), indicating that none of the other metal ions interfered with the detection of
Ac
Cu2+. The high selectivity may be due to its specific binding affinity of L toward Cu2+ over other metal ions. This result strongly indicated that receptor L is an excellent chemosensor for detection of Cu2+. The UV–visible titration experiment of L in the presence of Ni2+ was displayed in Fig. 10. The solution of L was colorless and had nearly no absorption at 525 nm. Upon addition of Ni2+, the solution changed from colorless to deep purple with a new 16
Page 17 of 44
absorption peak appearing at 525 nm. The significant spectra and color changes indicated that the free ligand had captured Ni2+. The absorbance gradually increased with the increasing of Ni2+ and was saturated at 0.5 equiv. of Ni2+, which mean the
ip t
binding mode was the same as L-Zn2+. In addition, Job’s plot analysis (Fig.S11, ESI†)
cr
as well as ESI mass spectra (Fig. S12, ESI†) confirmed the 2: 1 stoichiometry for L-
us
Ni2+ complex (Scheme 2). In the MS spectra, the formation of a 2 : 1 complex between L and Ni2+ was confirmed by the emergence of the peak at m/z = 777.5. The
an
linear response of L to Ni2+ based on the absorption spectra had also been exploited.
M
The detect limit was calculated to be 2.5 ×10-7 M (Fig. S13, ESI†). The results
ed
showed that L was a sensitive naked eyes indicator for Ni2+. In order to further evaluate the selectivity of L for Ni2+ sensing, competition
ce pt
experiments were carried out with other metal ions. The response of L to Ni2+ in the presence of 4 equiv. of various ions including Ba2+, K+, Na+, Li+, Al3+, Mg2+, Ca2+,
Ac
Pb2+, Sr2+, Cd2+, Hg2+, Fe2+, Zn2+, Cu2+, Mn2+ and Cu+ were shown in Fig. 11. Most competing metal ions show no interference with Ni2+ detection, except for Cu2+ and Fe2+. Therefore, L was an excellent naked eyes sensor for Ni2+ in aqueous medium. According to the result of competition experiments for Ni2+ detection by L, most heavy metal ions didn’t perturbed the recognition of Ni2+ (Fig. 11). However, Cu2+ will cause fatal interference. Namely, when both Cu2+ and Ni2+ were in the sample, 17
Page 18 of 44
the sensor would give priority to Cu2+ rather than Ni2+. As shown in Scheme 3, in the presence of both Cu2+ and Ni2+, the color of the sensor L was changed from colorless to yellow which was the color of L-Cu2+ complex. After adding cysteine, the color
ip t
changed from yellow to the color of L-Ni2+ purple.
cr
Cysteine with sulfydryl was the typical chelating agent for Cu2+, it was selected as
us
the mask for Cu2+ due to its good performance in previous work. 40 In the presence of cysteine, the absorption band at 470 nm caused by Cu2+ was completely disappeared,
an
and the color of L-Cu2+ was totally faded (Fig. 12). When both Cu2+ and Ni2+ were in
M
the sample, the absorption band was the same with the complex of L-Cu2+. With the
ed
addition of cysteine, the UV spectra changed fatally, the absorption peak at 470 nm was fade away and the new absorption band at 525 nm for L-Ni2+ rose up. Thus, the
ce pt
color of the solution was controlled by the competitive coordination of Cu2+ and Ni2+ with sensor L in the presence or absence of cysteine. Therefore, the existence of
Ac
cysteine was favorable for the selective detection of Ni2+ in the presence of Cu2+.
4. Conclusions
In summary, we have developed a highly effective metal sensor L, based on a Schiff base bearing an “O-N-N”-coordination site, for simultaneous detection of three transition metal ions (Zn2+, Ni2+ and Cu2+) through different signaling channels. This sensor showed a fluorescent “turn on” response to Zn2+ over other metal ions. Though 18
Page 19 of 44
the common disruptor Fe2+, Cu2+ and Ni2+ lead to fluorescence quenching, these free heavy metal ions are present at very low concentrations (nM) in cells and would not possibly interfere with the detection of Zn2+ in cell.41 Meanwhile, L was a
ip t
colorimetric sensor to Cu2+, Zn2+ and Ni2+, respectively. When both of Cu2+ and Ni2+
cr
existed in the same sample, L preferred to bind with Cu2+ rather than Ni2+. In the
us
presence of Cysteine, the sensor could be made selective towards Ni2+ over Cu2+. Cell imaging of confocal fluorescence microscopy further demonstrated that L can be used
an
for mapping Zn2+ levels in living cells. The study further illustrates a novel strategy to
M
differentiate the three transition metal ions (Cu2+, Zn2+ and Ni2+) by “naked eyes”.
ed
Thus, these results are significant and interesting for a new generation of metal recognition systems that can detect multiple analysts with one single small molecule
Ac
ce pt
without any complicated designs.
19
Page 20 of 44
Acknowledgment
This work was supported by the National Basic Research Program of China
ip t
(2012CB933800) and the National Natural Science Foundation of China (21374036). Reference
cr
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Wang, S. M. Jiang, Analyst 138 (2013) 2847–2857; (g) G. W. Men, C. R. Chen, S. T. Zhang, C. S. Liang, Y. Wang, M. Y. Deng, H. X. Shang, B. Yang S. M. Jiang, Dalton Trans. 44 (2015) 2755–2762. [33]. (a) A. Gogoi, S. Samanta and G. Das, Sensors and Actuators B 202 (2014) 788-
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[34]. B. D. jukic, P. A. Dube, F. Razavi, T. Seda, H. A. Jenkins, J. F. Britten, M. T. Lemaire, Inorg. Chem. 48 (2009) 699–707.
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[35]. A. Hens, P. Mondal, K. K. Rajak, Dalton Trans. 42 (2013) 14905–14915. [36]. Y. R. Li, J. Wu, X. J. Jin, J. W. Wang, S. Han, W.Y. Wu, J. Xu, W. S.Liu, X.
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[37]. (a) J. W. Liu and Y. Lu, J. Am. Chem. Soc. 129 (2007) 9838–9839; (b) W. Y.
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Lin, L. Yuan, Z. M. Cao, J. B. Feng, Y. M. Feng, Dyes and Pigments 83 (2009) 14–20. [38]. Y. Q. Tan, M. Liu, J. K. Gao, J. C. Yu, Y. J. Cui, Y. Yang, G. D. Qian, Dalton
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[39]. M. A. R. Meier and U. S. Schubert, Chem. Commun. (2005) 4610-4612. [40]. (a) W. H. Hao, A. McBride, S. McBride, J. P. Gao, Z. Y. Wang, J. Mater. Chem.
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21 (2011) 1040–1048; (b) R. K. Pathak, J. Dessingou, C. P. Rao, Anal. Chem. 84 (2012) 8294−8300; (c) Y. Hu, C. H. Heo, G. Kim, E. J. Jun, J. Yin, H. M. Kim and J. Yoon, Anal. Chem. 87 (2015) 3308 −3313. [41]. (a) G. Pigino, M. Migliorini, E. Paccagnini, F. Bernini, Ecotoxicology and Environmental Safety 64 (2006) 257–263; (b) A. R. Kay and S. M. Rumschik, Metallomics 3 (2011) 829–837; (c) M. R. Riley, D. E. Boesewetter, A. M. Kim, F. P. Sirvent, Toxicology 190 (2003) 171–184. 24
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Author Biographies Chunrong Chen received her BSc degree in chemistry from Sichuan University in 2013. Currently she is a graduate student of the College of Chemistry in Jilin
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University.
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Guangwen Men received his PhD degree in chemistry from Jilin University,
Bu received
his
BSc
degree
in
School
of
Stomatology
M
Wenhuan
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Changchun, China, in 2014. Currently he is a post doctor at Jilin University.
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from Jilin University in 2014. Currently he is a graduate student of the School of
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Stomatology in Jilin University.
Chunshuang Liang received her BSc degree in chemistry from Jilin University in
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2012. Currently she is a graduate student of the College of Chemistry in Jilin University.
Hongchen Sun is a professor of School of Stomatology at Jilin University. He received his PhD degree from Norman Bethune University of Medical Sciences in 1998. His main research interests are hard tissue remodeling and regeneration, 25
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preparation and application of biomaterials for bone regeneration.
Shimei Jiang is a professor of Chemistry at Jilin University. She received her PhD
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responsive supramolecular systems and nano-materials.
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degree from Jilin University in 1998. Her main research interests are stimuli-
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Figure captions:
Scheme 1 Synthetic route of compound L. Fig. 1 Fluorescence spectra (λex = 468 nm) (a) and bar graph (b) of L (50 μM) before
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and after addition of metal ions of (200 μM) Zn2+, Ba2+, K+, Na+, Li+, Al3+, Mg2+, Ca2+, Pb2+, Sr2+, Cd2+, Hg2+, Fe2+, Cu2+, Mn2+, Cu+ and Ni2+ in H2O-EtOH (8 : 2, v/v).
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The photos of L and L+Zn2+ were taken under UV light.
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Fig. 2 Fluorescence spectra of L (50 μM, λex = 468 nm) upon the addition of
increasing amounts of Zn2+ ions (0, 0.5, 1, 1.5, 3, 4, 5, 10, 15, 20, 25, 30, and 40 μM)
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in H2O-EtOH (8 : 2, v/v). The arrow indicates the change in the emission intensity with the increased Zn2+ ions. Inset: intensity at 584 nm versus the concentration of
M
Zn2+ added.
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Fig. 3 UV-vis spectra of L (50 μM) upon the addition of zinc ions (0, 0.5, 1, 1.5, 3, 4, 5, 6, 8, 10, 25, and 50μM) in H2O-EtOH (8 : 2, v/v). The arrow indicates the change
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in the absorbance with the increased Zn2+ ions. Inset: absorbance at 500 nm versus the concentration of Zn2+ added.
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Fig. 4 1H NMR of L and L-Zn2+ in DMSO-d6 : a) L-Zn2+, b) only L. Scheme 2 Proposed structures of L-M2+ complexes (M = Zn, Cu and Ni). Fig. 5 Fluorescence responses of L and L-Zn2+ at various ranges of pH in aqueous solution (H2O: EtOH = 8 : 2, v/v). λex= 468 nm. Fig. 6 Fluorescence images of Hela cell incubated with L and Zn2+. Cells were incubated with 50 μM L for 30 min (A and B) and then further with 25 μM ZnCl2 for 30 min (C and D). The left images (A, C) were observed with the light microscope 27
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and the right images (B, D) were taken with a fluorescence microscope. The scale bar is 50 μm. Fig. 7 (a) Absorption spectra of L (50 μM) in the presence of 200 μM various metal ions in aqueous solution (H2O: EtOH = 8 : 2, v/v). (b) The color of L (50 μM) upon
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addition of various metal ions .
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Fig. 8 UV-vis spectra of L (50 μM) upon the addition of Cu2+ (0, 1, 4, 6, 7, 10, 12, 14, 16, 18, 20, 22, 25, and 30 μM) in aqueous solution (H2O: EtOH = 8 : 2, v/v). The
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arrow indicates the change in the absorbance with the increased Cu2+ ions. Inset:
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absorbance at 470 nm versus the concentration of Cu2+ added.
Fig. 9 Effect of competitive metal ions (25 μM) on the interaction between sensor L
M
(50 μM) and Cu2+ ion (25 μM) in aqueous solution (H2O: EtOH = 8 : 2, v/v).
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Fig. 10 UV–vis spectra of L (50 μM) upon the addition of Ni2+ (0, 1, 2, 3, 4, 5, 6, 8, 10, 15, 18, 20, and 25 μM) in aqueous solution (H2O: EtOH = 8 : 2, v/v). The arrow
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indicates the change in the absorbance with the increased Ni2+ ions. Inset: absorbance at 525 nm versus the concentration of Ni2+ added. Fig. 11 Effect of competitive metal ions (25 μM) on the interaction between sensor L
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(50 μM) and Ni2+ ion (25 μM) in aqueous solution (H2O: EtOH = 8 : 2, v/v). Scheme 3 Competitive coordination pattern of Cu2+ and Ni2+ with L. Fig. 12 UV–vis spectra of competitive coordination of Cu2+and Ni2+ with L in the presence and absence of cysteine in aqueous solution (H2O: EtOH = 8 : 2, v/v).
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Scheme 1 Synthetic route of compound L.
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Fig. 1 Fluorescence spectra (λex = 468 nm) (a) and bar graph (b) of L (50 μM) before and after addition of metal ions of (200 μM) Zn2+, Ba2+, K+, Na+, Li+, Al3+, Mg2+, Ca2+, Pb2+, Sr2+, Cd2+, Hg2+, Fe2+, Cu2+, Mn2+, Cu+ and Ni2+ in H2O-EtOH (8 : 2, v/v).
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M
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The photos of L and L+Zn2+ were taken under UV light.
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Fig. 2 Fluorescence spectra of L (50 μM, λex = 468 nm) upon the addition of increasing amounts of Zn2+ ions (0, 0.5, 1, 1.5, 3, 4, 5, 10, 15, 20, 25, 30, and 40μM) in H2O-EtOH (8 : 2, v/v). The arrow indicates the change in the emission intensity
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with the increased Zn2+ ions. Inset: intensity at 584 nm versus the concentration of
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Zn2+ added.
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Fig. 3 UV-vis spectra of L (50 μM) upon the addition of zinc ions (0, 0.5, 1, 1.5, 3, 4, 5, 6, 8, 10, 25, and 50μM) in H2O-EtOH (8 : 2, v/v). The arrow indicates the change in the absorbance with the increased Zn2+ ions. Inset: absorbance at 500 nm versus the
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M
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concentration of Zn2+ added.
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Fig. 4 1H NMR of L and L-Zn2+ in DMSO-d6 : a) L-Zn2+, b) only L.
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Scheme 2 Proposed structures of L-M2+ complexes (M = Zn, Cu and Ni).
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Fig. 5 Fluorescence responses of L and L-Zn2+ at various ranges of pH in aqueous
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M
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solution (H2O: EtOH = 8 : 2, v/v). λex= 468 nm.
35
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Fig. 6 Fluorescence images of Hela cell incubated with L and Zn2+. Cells were incubated with 50 μM L for 30 min (A and B) and then further with 25 μM ZnCl2 for 30 min (C and D). The left images (A, C) were observed with the light microscope and the right images (B, D) were taken with a fluorescence microscope. The scale bar
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is 50 μm.
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Fig. 7 (a) Absorption spectra of L (50 μM) in the presence of 200 μM various metal ions in aqueous solution (H2O: EtOH = 8 : 2, v/v). (b) The color of L (50 μM) upon
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addition of various metal ions .
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Fig. 8 UV-vis spectra of L (50 μM) upon the addition of Cu2+ (0, 1, 4, 6, 7, 10, 12, 14, 16, 18, 20, 22, 25, and 30 μM) in aqueous solution (H2O: EtOH = 8 : 2, v/v). The arrow indicates the change in the absorbance with the increased Cu2+ ions. Inset:
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absorbance at 470 nm versus the concentration of Cu2+ added.
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Fig. 9 Effect of competitive metal ions (25 μM) on the interaction between sensor L
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M
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(50 μM) and Cu2+ ion (25 μM) in aqueous solution (H2O: EtOH = 8 : 2, v/v).
39
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Fig. 10 UV–vis spectra of L (50 μM) upon the addition of Ni2+ (0, 1, 2, 3, 4, 5, 6, 8, 10, 15, 18, 20, and 25 μM) in aqueous solution (H2O: EtOH = 8 : 2, v/v). The arrow indicates the change in the absorbance with the increased Ni2+ ions. Inset: absorbance
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ed
M
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at 525 nm versus the concentration of Ni2+ added.
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Fig. 11 Effect of competitive metal ions (25 μM) on the interaction between sensor L
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M
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(50 μM) and Ni2+ ion (25 μM) in aqueous solution (H2O: EtOH = 8 : 2, v/v).
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Scheme 3 Competitive coordination pattern of Cu2+ and Ni2+ with L.
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Fig. 12 UV–vis spectra of competitive coordination of Cu2+and Ni2+ with L in the
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presence and absence of cysteine in aqueous solution (H2O: EtOH = 8 : 2, v/v).
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