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Europium(III) complex-functionalized magnetic nanoparticle as a chemosensor for ultrasensitive detection and removal of copper(II) from aqueous solution† Jing Liu,a Wei Zuo,a Wei Zhang,b Jian Liu,a Zhiyi Wang,a Zhengyin Yanga and Baodui Wang*a Ultrasensitive, accurate detection and separation of heavy metal ions is very important in environmental monitoring and biological detection. In this paper, a highly sensitive and specific detection method for
Received 20th June 2014 Accepted 1st August 2014
Cu2+ based on the fluorescence quenching of a europium(III) hybrid magnetic nanoprobe is presented. This nanoprobe can detect Cu2+ over a wide pH range (5.0–10.0) with a detection limit as low as 0.1 nM
DOI: 10.1039/c4nr03454f
and it can be used for detecting Cu2+ in living cells. After the magnetic separation, the Cu2+ concentration decreased to 1.18 ppm, which is less than the US EPA drinking water standard (1.3 ppm),
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and more than 70% Cu2+ could be removed when the amount of nanocomposite 1 reached 1 mg.
Introduction Nowadays, the design of multi-functional nanoprobes that can accurately and selectively recognize and rapidly remove heavy metal ions in organisms and the environment is particularly important, because human health and the environment may have been seriously affected by these metal ions.1,2 Copper maintains enzymatic biological processes and is the third most abundant essential trace element in the human body. On the other hand, it is also commonly found in contaminated water3,4 and high exposures to Cu2+ not only have serious effects on humans, resulting in neurodegenerative diseases such as Wilson's disease, Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis, gastrointestinal disorders and kidney damage, but they can also hinder the self-purication capability of seas and rivers by chemical and biological degradation.5,6 The US EPA (Environmental Protection Agency) has set the maximum permissible content of copper in drinking water to be 1.3 ppm (20 mM). Therefore, the sensitive sensing and accurate monitoring of copper ions is important and warrants further research. As detailed in previous literature reports, many chemosensors based on atomic absorption/ emission spectroscopy,7 inductively coupled plasma mass spectrometry8 and electrochemical methods9 have been a
Key Laboratory of Nonferrous Metal Chemistry and Resources Utilization of Gansu Province and State Key Laboratory of Applied Organic Chemistry Lanzhou University Gansu, Lanzhou University, Lanzhou 730000, P.R. China. E-mail: [email protected]
b
Key Laboratory of Preclinical Study for New Drugs of Gansu Province, School of Basic Medical Sciences, Lanzhou University, Lanzhou 730000, P.R. China
† Electronic supplementary information (ESI) available: Scheme S1, Fig. S1–S10, Tables S1–S4. See DOI: 10.1039/c4nr03454f
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developed. However, all of these methods require expensive instruments and complicated sample pretreatment, which limits their wide and routine application. Fluorescence dosimetric detection becomes a practical and economic method because of its high sensitivity, selectivity, simplicity and real-timing monitoring with fast response time.10,11 In recent years, although numerous uorescent dyebased sensors for Cu2+ ions have been developed,12,13 these chemosensors inevitably have some problems, such as poor water solubility, low detection sensitivity and easy photobleaching. Compared to traditional organic dyes, lanthanide complexes possess some superior photophysical properties in terms of a large Stokes shi, narrow emission bands, long luminescence lifetimes and high quantum yields or photostability.14,15 These advantages make lanthanide complexes attractive probes for the uorescent sensing of metal ions,16,17 anions18,19 or biomolecules.20 On the other hand, separation and removal of probes and heavy metal ions remains an issue that needs to be solved for sensing and biotechnological applications. Numerous techniques, such as magnetic separation, chemical precipitation21 and physical absorption,22 have recently been developed for heavy metal ion enrichment and isolation. Among these, magnetic separation is an extensively applied and extremely promising approach for metal ion separation because of its simple manipulation and high capture efficiency. In addition, uorescent–magnetic nanoparticles (NPs) are ideal candidates for metal ion recognition and separation in the development of sensing systems23,24 because they possess superparamagnetism,25 good biocompatibility26 and the ability for uorescent signal amplication. So, the incorporation of
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Schematic description of Cu2+ detection and removal using a europium complex functionalized-Fe3O4 nanoparticle (1). Scheme 1
Fig. 1 TEM images of (A) the as-synthesized Fe3O4 NPs from a hexane dispersion and (B) Fe3O4-DBA-PEG-NH-DTPA-AMC:Eu3+ (1) from a water dispersion.
magnetic nanomaterials (Fe3O4) into lanthanide complex-based uorescent sensors is a promising way for detection and separation of toxic metal ions from various environments.23,27–29 In this paper, a luminescent europium complex hybrid Fe3O4 nanoparticle (abbreviated as Fe3O4-DBA-PEG-NH-DTPAAMC:Eu3+; Scheme 1) that could not only detect Cu2+ in aqueous solution and in living cells but can also enrich and remove Cu2+ in water, is reported. The detection of Cu2+ by this nanoprobe is illustrated in Scheme 1. The detection method based on 1 shows a good sensitivity and specicity for Cu2+, and the detection limit is 0.1 nM (6.35 ppt). In addition, because the probe contains superparamagnetic Fe3O4 NPs, this nanoprobe can be easily separated from the solution by using a magnet aer each measurement.
measured at different time intervals with different Cu2+ concentrations. The interaction between 1 and the Cu2+ ions is rapid because the uorescence quenching equilibrium can be reached within 5 min (Fig. S2†). The effects of pH on detection were investigated by dispersing the sensor 1 into solutions with different pH values and the same concentration of Cu2+ ions (10 mM). As shown in the Fig. S3,† the uorescence intensity of the detection reaction for Cu2+ were stable over a wide range of pH (from 5 to 10), indicating that using sensor 1 for the detection of Cu2+ has potential applications under some physiological and environmental conditions. In this work, the detection of Cu2+ was carried out in aqueous solution with a neutral pH.
Results and discussion
The uorescence spectroscopy of nanoprobe 1 to Cu2+
Synthesis and characterization of nanoprobe 1 3+
The Fe3O4 NPs and Fe3O4-DBA-PEG-NH-DTPA-AMC:Eu nanoprobe (1) were synthesized using a previous method.30,31 The uorescent nanoprobe consists of magnetic Fe3O4 NPs modied by polyethylene glycol-3,4-dihydroxybenzylamine (DBA-PEG-NH2) and Eu3+ ions integrated with a polyaminocarboxylate-based chelator (diethylene triamine pentaacetic acid DTPA). The organic chromophore, 7-amino-4-methyl coumarin (AMC) acted as an antenna absorbing excitation light and as a donor transferring energy to the Eu3+. According to potentiometric studies,32 the DTPA-AMC has a higher binding constant for Cu2+ (K ¼ 1021.77) than Eu3+ (K ¼ 1019.67, see ESI, Fig. S1, Tables S1 and S2†). Consequently, in the absence of Cu2+, this lanthanide–NPs complex emits the characteristic red uorescence of Eu3+. In the presence of Cu2+, Eu3+ can be replaced by Cu2+ to form Fe3O4-DBA-PEG-NH-DTPA-AMC:Cu2+ (2), which results in the uorescent quenching of 1. The transmission electron microscopy (TEM) images indicate that the morphology of as-synthesized Fe3O4 NPs and nanoprobe 1 were similar, and 1 could easily disperse in water (Fig. 1A and B). Effects of time and pH on the nanoprobe 1 To understand the quenching rate of Fe3O4-DBA-PEG-NHDTPA-AMC:Eu3+ by Cu2+, the uorescent intensity of 1 was
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The sensing ability of 1 for Cu2+ was investigated in an aqueous solution of Tris–HCl buffer (50 mM, pH 7.20) using uorescence emission spectroscopy. As shown in Fig. 2A, upon excitation at 325 nm, the solution of 1 showed the emission of europium at 580 nm, 595 nm and 616 nm via an intramolecular energy transfer from AMC to Eu3+. However, upon addition of Cu2+ at concentrations from 1 mM to 200 mM, the emission intensity of 1 gradually decreased, indicating that Eu3+ was replaced by Cu2+. Consequently, the emission color of the probe showed a dramatic change from red to colorless in the presence of Cu2+ (Fig. S4†). Also, the change between the uorescent intensities
Fig. 2 (A) Fluorescent emission spectra of 10 mM 1 upon addition of Cu2+ at concentrations from 1 mM to 200 mM in Tris–HCl buffer (50 mM, pH 7.20). (B) The linear relationship between the fluorescent intensity of 10 mM of 1 and Cu2+ concentrations from 0.1 nM to 1 nM at 616 nm.
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of 1 and the concentrations of Cu2+ in the range 0.1–1 nM shows a good linear correlation (Fig. 2B). The detection limit (DL) based on the formation of 2 is 6.35 ppt. This DL for Cu2+ is far less than DLs obtained with other probes based on organic uorophores13,33 and is sufficiently lower than the acceptable limit of the US EPA value of Cu2+ ions (1.3 ppm) in drinking water, indicating that the probe 1 is sensitive for detecting Cu2+ in aqueous solutions. Selectivity of nanoprobe 1 for Cu2+ To check whether nanoprobe 1 can selectively detect Cu2+, the effects of other metal ions on the uorescence of 1 have also been examined to evaluate the selectivity under the same experimental conditions. As shown in Fig. 3A (red bars), only the addition of Cu2+ resulted in a great uorescent intensity reduction from 117 a.u. to 30 a.u. at 616 nm, and the uorescent color of the probe changed from red to colorless. In contrast, the addition of large excesses of other metal ions, such as: Ag+, Al3+, Ba2+, Ca2+, Cd2+, Co2+, Cr3+, Fe2+, Fe3+, Hg2+, K+, Li+, Mg2+, Mn2+, Na+, Ni2+, Pb2+ and Zn2+, caused only slight changes, which were easily observed by the naked eye with irradiation from a UV-lamp (Fig. S5†). Therefore, nanoprobe 1 displayed high selectivity for the luminescent sensing of Cu2+. To further explore the use of nanoprobe 1 as an ion-selective nanosensor for Cu2+, competitive experiments were performed by adding Cu2+ to solutions of 1 in the presence of other cations. As shown in Fig. 3A (blue bars), signicant quenching of uorescence was observed for 1 upon the addition of Cu2+ in the presence of other metal ions. The results indicate that the sensing of Cu2+ by 1 is hardly disturbed by these commonly coexisting ions. Therefore, 1 can act as a highly selective probe for Cu2+. Quenching mechanism of Cu2+ In order to further understand the quenching mechanism of Cu2+ with 1, uorescence lifetime and the characteristic features of the uorescence intensity-based Stern–Volmer plot of 1 in the presence and absence of Cu2+ were investigated. As shown in Fig. S6 and Table S3,† the luminescent lifetimes of complexes 1 and 2 were measured by tting them to
Fig. 3 Effects of various metal ions on the fluorescent intensity of 10 mM of 1 in Tris–HCl buffer (50 mM, pH 7.20). The red bars represent the emission intensity of 1 in the presence of interfering ions (100 mM) at 616 nm. The blue bars represent the emission intensity of 1 upon addition of Cu2+ (100 mM) in the presence of each interfering metal ion (100 mM).
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biexponential decay curves. In the absence of Cu2+, biexponential behavior was observed with s values (relative weighting) of 0.56 ms (56%) and 1.16 ms (44%). In the presence of 7 mM Cu2+, the corresponding values were 0.41 ms (51%) and 1.06 ms (49%). Upon the addition of Cu2+, the average emission lifetime was reduced from 0.83 to 0.73 ms, indicating that excited-state molecules of 1 interacted with Cu2+. Fig. S7† shows the intensity-based Stern–Volmer plot of F0/F versus [Cu2+]. Interestingly, F0/F versus [Cu2+] plots show a distinct upwards curvature, implying that the 1–Cu2+ uorophore–quencher pair in solution does not show simple Stern–Volmer behavior20,34 and the static quenching and dynamic quenching occur concomitantly in this case. The static quenching is the predominant mechanism because the F0/F ¼ 1.51 is a higher value than hs0i/hsi ¼ 1.13 at 7 mM Cu2+. hs0i and hsi are average lifetimes of 1 in the absence and presence of Cu2+, respectively. Considering the following equations: 2 F0 ¼ 1 þ ðKD þ KS Þ Cu2þ þ KD KS Cu2þ F
(1)
F0 ¼ 1 þ KD Cu2þ 1 þ KS Cu2þ F
(2)
F0 ¼ 1 þ Kapp Cu2þ F
(3)
Kapp ¼
F0 1 1 ¼ ðKD þ KS Þ þ KD KS Cu2þ ½Cu2þ F KS2 KSI + S ¼ 0
(4)
(5)
The apparent quenching constant is calculated at each quenching concentration. A plot of Kapp versus [Cu2+] yields a straight line with an intercept of KD + KS and a slope of KDKS. The individual values can be obtained from the two solutions of the quadratic equation. The KD and KS were found to be 2.62 103 M1 and 7.65 103 M1, respectively. Separation of Cu2+ in water Magnetic separation is regarded as one of the most promising methods for Cu2+ removal from water. An extremely sensitive method based on uorescent spectroscopy was employed to detect free copper ions aer the magnetic separation, and then the separation efficiency was evaluated. Because 1 exhibits superparamagnetism at room temperature (Fig. S8†), we attempted to use 2 formed by adding Cu2+ to 1 for indirectly separating the Cu2+ from the aqueous solution. The different amounts of nanocomposite 1 (0, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4 mg) were added into 2 mL of a Cu2+ (4 ppm) aqueous solution, and then the mixture was gently stirred for 1 h at room temperature. Aer reaction, the hybrid formed, 2, containing Cu2+ was enriched and separated using a magnet, and the supernatant solution was collected for the further evaluation of the residual Cu2+ concentrations and the separation efficiency. The separation efficiency was dened as the decrease in the Cu2+ concentration before and aer magnetic separation. There was a quasiquantitative relationship between the Cu2+ concentration and
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Fig. 4 (A) The residual Cu2+ concentrations in aqueous solution after magnetic separation using different amounts of 1 (0, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4 mg). (B) Removal efficiency using different amounts of 1 for removal of Cu2+ in aqueous solution.
the uorescent intensity of nanoprobe 1. Firstly, the uorescent intensity of the standard compound AMC-DTPA-NH-PEGDBA:Eu3+ (105 M) with and without the addition of Cu2+ (4 ppm) standard solution was set as F0 and F, and DFS was dened as the value of the difference between F0 and F. Therefore, DFS acting as the reference standard was used to estimate the residual Cu2+ concentrations and the separation efficiency of the following procedures. Secondly, the Cu2+ concentration in the supernatant solution containing different amounts of nanoprobe 1 aer magnetic separation was indirectly detected by using the standard compound AMC-DTPA-NH-PEGDBA:Eu3+ (105 M), where the uorescent intensity changes were set as DFx. Finally, the results from the previous two steps were compared to estimate the approximate Cu2+ concentration and to calculate the separation efficiency. In addition, every Cu2+ concentration in the supernatant solution was prepared from a Cu2+ standard concentration and again veried by using the standard compound AMC-DTPA-NH-PEG-DBA:Eu3+ (Fig. S9 and Table S4†). As shown in Fig. 4A, when the dispersion amount of 1 in water reaches 1 mg, the concentration of Cu2+ decreases to 1.18 ppm, which is less than the US EPA drinking water standard (1.3 ppm). The investigation of Cu2+ separation efficiency was carried out as shown in Fig. 4B. By comparing the Cu2+ concentration before and aer the magnetic separation, more than 70% of Cu2+ was removed during the 1 h collection which corresponded to 1 mg of nanocomposite 1. The Eu3+ ions exchanged in solution could be detected and were removed by the Fe3O4-DBA-PEG-NH-DTPA-AMC (Fig. S10†). These experimental results demonstrated that 1 could not only be used as a detection reagent for Cu2+, but could also be used as a water purication agent to remove any excess copper ions.
Fluorescence imaging The ability of nanocomposite 1 to detect Cu2+ within living cells was also evaluated by using laser confocal uorescence imaging. Under selective excitation at 325 nm, HeLa cells incubated with nanocomposite 1 (10 mM Eu3+) for 1 h at 37 C showed the characteristic red emission uorescence of europium (Fig. 5A). When the nanocomposite 1-loaded cells were incubated with 35 mM Cu2+ for another hour, the red uorescence was almost quenched (Fig. 5B). This implies that
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Fluorescent images of HeLa cells (A) after incubation with 1 for 1 h and (B) after addition of 35 mM Cu2+ to the 1 treated cells for 1 h. Excitation at 325 nm and emission at 616 nm. Fig. 5
nanocomposite 1 could be used to monitor Cu2+ in living cells.
Conclusion In conclusion, lanthanide-functionalized magnetic nanoparticles were prepared that act as a new type of uorescent sensor for ultrasensitive sensing and efficiently separating Cu2+ in aqueous solution. These multi-functional nanoparticles exhibit a high sensitivity and selectivity for targeting Cu2+ ions over various other metal ions, with a detection limit of 0.1 nM. Aer interaction with Cu2+, the characteristic red uorescence of europium was quenched and the uorescent color changed to colorless. The sensor also exhibited high selectivity for Cu2+ over other common metal ions. Moreover, 1 was shown to be stable in aqueous solution over a wide pH range and was suitable for uorescent imaging in living cells, which indicates that 1 has potential applications in biological analysis. Furthermore, copper ions could be collected and separated using these magnetic nanoprobes with a commercial magnet. The experimental results reported here open up a new method for simple and reliable monitoring of environmentally toxic ions as well as enrichment/removal of excess toxic ions.
Experimental Chemicals Iron(III) acetylacetonate, oleylamine, benzyl ether, 3,4-dihydroxybenzaldehyde, 7-amino-4-methyl coumarin (AMC), and PEG (MW ¼ 4000) were purchased from Sigma-Aldrich and were used without further purication. Diethylenetriaminepentaacetic acid dianhydride (DTPAA),35 1,u-diaminopolyoxyethylene (MW ¼ 4000),36 polyethylene glycol-3,4-dihydroxybenzylamine (H2N-PEG-DBA, MW ¼ 4000),27 Fe3O4 nanoparticles,30 and Fe3O4-DBA-PEG-NH-DTPA-AMC:Eu3+ (1)31 were synthesized according to the published method. The solution of metal ions (AgNO3, Al(NO3)3, BaCl2, Ca(NO3)2, Cd(NO3)2, Co(NO3)2, CrCl3, CuCl2, FeCl3, FeSO4, Hg(ClO4)2, KNO3, LiBr, Mg(NO3)2, Mn(CH3COO)2, NaNO3, Ni(NO3)2, Pb(CH3COO)2, Zn(NO3)2) were dissolved in ultrapure water. Aqueous Tris–HCl buffer (50 mM, pH 7.20, NaCl 50 mM) was used to maintain the pH value and the ionic strength of all the solutions used in the
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experiments. EuCl3$6H2O was obtained by dissolving Eu2O3 in hydrochloric acid followed by successive fuming to remove excess acid.
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Instrumentation The uorescence spectra were recorded on a Shimadzu RF-5301 spectrouorophotometer. The uorescence lifetimes were recorded on the Edinburgh Photonics FLS920 steady statetransient state uorescence spectrometer. The TEM measurements were carried out using a Philips EM 420 (120 kV). Fluorescent images were taken on Zeiss Leica inverted epiuorescence/reectance laser scanning confocal microscope. The concentrations of Eu3+ were determined by inductively-coupled plasma – atomic emission spectroscopy (ICPAES). The hysteresis loop was obtained at 300 K with a Lake Shore Cryotronics 7400 VSM system. Preparation of Fe3O4-DBA-PEG-NH-DTPA-AMC:Eu3+ (1) Fe3O4-DBA-PEG-NH-DTPA-AMC:Eu3+ (1) was synthesized according to the previously published method. Briey, 35.7 mg of DTPAA (0.1 mmol) and 17.5 mg AMC (0.1 mmol) were dissolved in 2 mL and 1 mL of anhydrous dimethylformamide (DMF), respectively. Dry triethylamine (100 mL) was added to the DTPAA solution. Then, the DMF solutions of DTPAA and AMC were mixed, and the mixture was stirred for 10 h at room temperature. Then 10 mL of a dry CH2Cl2 solution of 0.4 g DBAPEG-NH2 (0.1 mmol) was added to the DMF solution of DTPAAMC, and the mixture obtained was stirred for 24 h at room temperature. Then EuCl3$6H2O 36.7 mg (0.1 mmol) was added at an equivalent molar rate to the AMC-DTPA-NH-PEG-DBA solution for 12 h with stirring at room temperature. The product was precipitated by adding diethyl ether and collected by centrifugation. Aer washing with DMF, CH2Cl2 and diethyl ether, the product Eu3+:AMC-DTPA-NH-PEG-DBA was then dried under vacuum. The CHCl3 solution of Fe3O4 nanoparticles was added to 100 mg of the Eu(III) complex DMF solution and then stirred for 12 h at room temperature. The product was precipitated by adding petroleum ether, collected by centrifugation at 4000 rpm, washed with DMF, ethanol and petroleum ether, and then redispersed in water and dialysed with H2O for 24 h to remove unreacted organic molecules. 32
Potentiometric studies
The protonation constant of the ligand (DTPA-AMC) and the stability constant (i.e., binding constant) of the complexes (Eu:DTPA-AMC and Cu:DTPA-AMC) were studied at 25 C and at an ionic strength of I ¼ 0.10 mol dm3 (NaCl) using potentiometric pH titration. The protonation constant of the ligand was calculated rst and this was then used for determining the stability constant of the complexes. Potentiometric titrations were carried out using the ligand DTPA-AMC for calculating the protonation constants of the ligand. Then, the ligand DTPAAMC and two different metal solutions (Eu3+ and Cu2+) were used for calculating the stability constant of the metal complexes. Each of the potentiometric measurements were performed by titrating 50 mL of the aqueous NaCl solutions
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against the standard NaOH solutions until the formation complexes were stable. The overall experimental procedure involved the potentiometric titrations of the following solutions: (a) 10 mL of 1 mM ligands + 50 mL of 0.1 M NaCl (for the determination of the protonation constant of ligand), (b) 10 mL of 1 mM ligands + 10 mL of 1 mM metal ion + 50 mL of 0.1 M NaCl (for the determination of the stability constant of complexes). The computations of protonation constant of ligand and the stability constant of complexes from potentiometric data were carried out using the Bjerrun's Half-n Method. All of the potentiometric data were recorded between pH 3.0 and 10.0. Fluorescence spectral studies Stock solutions of various metal ions were prepared in ultrapure water. A stock solution of 1 (0.5 mM Eu3+) was analysed by ICPAES in pure water. The solution of 1 was then diluted to 10 mM with Tris–HCl (50 mM, pH 7.20). In titration experiments, a 2 mL solution of 1 (10 mM) was placed in to a quartz optical cell with a 1 cm optical path length, and the Cu2+ solutions from 1 mM to 200 mM, were gradually added into the quartz optical cell using a micropipette. In selectivity experiments, the test samples were prepared by using appropriate amounts of the metal ion solution. Finally, the concentration of solution 1 was 10 mM and the metal ion concentration was 100 mM. To study the coexistence of Cu2+ and other metal ions studies, 100 mM Cu2+ was then added to the solutions of 1 and metal ions. In reaction time experiments, the uorescent intensity of 10 mM 1 was measured at different time intervals in the presence of 1 mM, 10 mM and 100 mM Cu2+ in an aqueous solution of Tris–HCl. In pH effect experiments, the uorescent intensity of 10 mM of 1 was measured without and with 10 mM Cu2+ at different pH values. The pH values were regulated in the range of 3 to 11 using diluted hydrochloric acid or sodium hydroxide. For uorescence measurements, excitation was at 325 nm, and emission was from 560 to 640 nm. There are three special peaks at 580 nm, 595 nm, 616 nm and the emission intensity at 616 nm was recorded in some experiments. Removal of Cu2+ by Fe3O4-DBA-PEG-NH-DTPA-AMC:Eu3+ (1) Preparation of standard samples. The Eu3+:AMC-DTPA-NHPEG-DBA standard solution were prepared to give a concentration of 10 mM Eu3+. Aer addition of CuCl2 solution (4 mg L1), the uorescence intensity was measured. Different amounts of 1 from 0, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4 mg were dispersed into 2 m of CuCl2 solution (4 mg L1). Aer stirring the mixture for about 1 h, the dispersed substance was separated using an external magnetic eld and centrifugation for separation of the mixture using precipitation, and then the residual concentrations of Cu2+ in water were measured by comparison with a 10 mM Eu3+:AMC-DTPA-NH-PEG-DBA standard solution. Lifetime The lifetime decay curves were recorded on the FLS920 steady state-transient state uorescence spectrometer. Excitation was Nanoscale, 2014, 6, 11473–11478 | 11477
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at 325 nm, and emission was at 616 nm. The lifetimes (s) of 1 (50 mM, s0) and 7 mM Cu2+ reactions with 1 (50 mM) formed nanocomposite 2 (s) were measured, respectively. The average lifetimes hs0i (1), hsi (2) were calculated using the equation hsi ¼ P 2P Aisi / Aisi.37
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Fluorescence microscopic imaging HeLa cells were grown in Dulbecco's modied Eagle medium supplemented with 10% fetal bovine serum in an atmosphere of 5% CO2 and 95% humidied air at 37 C for 24 h. Then the cells were treated with the various compounds using uorescence microscopic imaging. The cultured cells were treated with 1 and incubated for 1 h. Subsequently, the cells were treated with 35 mM CuCl2 for another 1 h. Aer incubation for the corresponding time, the cells were washed three times with phosphate buffered saline to remove free compounds and ions before analysis. Then uorescence microscopic images were acquired. All confocal images were collected with a Zeiss Leica inverted epiuorescence/reectance laser scanning confocal microscope with excitation at 325 nm and emission at 616 nm.
Acknowledgements The work was supported by the National Natural Science Foundation of China (21271093), the NCET (13-0262), and the Fundamental Research Funds for the Central Universities (lzujbky-2013-56 and lzujbky-2014-k06).
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PAPER
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Europium(III) complex-functionalized magnetic nanoparticle as a chemosensor for ultrasensitive detection and removal of copper(II) from aqueous solution† Jing Liu,a Wei Zuo,a Wei Zhang,b Jian Liu,a Zhiyi Wang,a Zhengyin Yanga and Baodui Wang*a Ultrasensitive, accurate detection and separation of heavy metal ions is very important in environmental monitoring and biological detection. In this paper, a highly sensitive and specific detection method for
Received 20th June 2014 Accepted 1st August 2014
Cu2+ based on the fluorescence quenching of a europium(III) hybrid magnetic nanoprobe is presented. This nanoprobe can detect Cu2+ over a wide pH range (5.0–10.0) with a detection limit as low as 0.1 nM
DOI: 10.1039/c4nr03454f
and it can be used for detecting Cu2+ in living cells. After the magnetic separation, the Cu2+ concentration decreased to 1.18 ppm, which is less than the US EPA drinking water standard (1.3 ppm),
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and more than 70% Cu2+ could be removed when the amount of nanocomposite 1 reached 1 mg.
Introduction Nowadays, the design of multi-functional nanoprobes that can accurately and selectively recognize and rapidly remove heavy metal ions in organisms and the environment is particularly important, because human health and the environment may have been seriously affected by these metal ions.1,2 Copper maintains enzymatic biological processes and is the third most abundant essential trace element in the human body. On the other hand, it is also commonly found in contaminated water3,4 and high exposures to Cu2+ not only have serious effects on humans, resulting in neurodegenerative diseases such as Wilson's disease, Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis, gastrointestinal disorders and kidney damage, but they can also hinder the self-purication capability of seas and rivers by chemical and biological degradation.5,6 The US EPA (Environmental Protection Agency) has set the maximum permissible content of copper in drinking water to be 1.3 ppm (20 mM). Therefore, the sensitive sensing and accurate monitoring of copper ions is important and warrants further research. As detailed in previous literature reports, many chemosensors based on atomic absorption/ emission spectroscopy,7 inductively coupled plasma mass spectrometry8 and electrochemical methods9 have been a
Key Laboratory of Nonferrous Metal Chemistry and Resources Utilization of Gansu Province and State Key Laboratory of Applied Organic Chemistry Lanzhou University Gansu, Lanzhou University, Lanzhou 730000, P.R. China. E-mail: [email protected]
b
Key Laboratory of Preclinical Study for New Drugs of Gansu Province, School of Basic Medical Sciences, Lanzhou University, Lanzhou 730000, P.R. China
† Electronic supplementary information (ESI) available: Scheme S1, Fig. S1–S10, Tables S1–S4. See DOI: 10.1039/c4nr03454f
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developed. However, all of these methods require expensive instruments and complicated sample pretreatment, which limits their wide and routine application. Fluorescence dosimetric detection becomes a practical and economic method because of its high sensitivity, selectivity, simplicity and real-timing monitoring with fast response time.10,11 In recent years, although numerous uorescent dyebased sensors for Cu2+ ions have been developed,12,13 these chemosensors inevitably have some problems, such as poor water solubility, low detection sensitivity and easy photobleaching. Compared to traditional organic dyes, lanthanide complexes possess some superior photophysical properties in terms of a large Stokes shi, narrow emission bands, long luminescence lifetimes and high quantum yields or photostability.14,15 These advantages make lanthanide complexes attractive probes for the uorescent sensing of metal ions,16,17 anions18,19 or biomolecules.20 On the other hand, separation and removal of probes and heavy metal ions remains an issue that needs to be solved for sensing and biotechnological applications. Numerous techniques, such as magnetic separation, chemical precipitation21 and physical absorption,22 have recently been developed for heavy metal ion enrichment and isolation. Among these, magnetic separation is an extensively applied and extremely promising approach for metal ion separation because of its simple manipulation and high capture efficiency. In addition, uorescent–magnetic nanoparticles (NPs) are ideal candidates for metal ion recognition and separation in the development of sensing systems23,24 because they possess superparamagnetism,25 good biocompatibility26 and the ability for uorescent signal amplication. So, the incorporation of
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Paper
Schematic description of Cu2+ detection and removal using a europium complex functionalized-Fe3O4 nanoparticle (1). Scheme 1
Fig. 1 TEM images of (A) the as-synthesized Fe3O4 NPs from a hexane dispersion and (B) Fe3O4-DBA-PEG-NH-DTPA-AMC:Eu3+ (1) from a water dispersion.
magnetic nanomaterials (Fe3O4) into lanthanide complex-based uorescent sensors is a promising way for detection and separation of toxic metal ions from various environments.23,27–29 In this paper, a luminescent europium complex hybrid Fe3O4 nanoparticle (abbreviated as Fe3O4-DBA-PEG-NH-DTPAAMC:Eu3+; Scheme 1) that could not only detect Cu2+ in aqueous solution and in living cells but can also enrich and remove Cu2+ in water, is reported. The detection of Cu2+ by this nanoprobe is illustrated in Scheme 1. The detection method based on 1 shows a good sensitivity and specicity for Cu2+, and the detection limit is 0.1 nM (6.35 ppt). In addition, because the probe contains superparamagnetic Fe3O4 NPs, this nanoprobe can be easily separated from the solution by using a magnet aer each measurement.
measured at different time intervals with different Cu2+ concentrations. The interaction between 1 and the Cu2+ ions is rapid because the uorescence quenching equilibrium can be reached within 5 min (Fig. S2†). The effects of pH on detection were investigated by dispersing the sensor 1 into solutions with different pH values and the same concentration of Cu2+ ions (10 mM). As shown in the Fig. S3,† the uorescence intensity of the detection reaction for Cu2+ were stable over a wide range of pH (from 5 to 10), indicating that using sensor 1 for the detection of Cu2+ has potential applications under some physiological and environmental conditions. In this work, the detection of Cu2+ was carried out in aqueous solution with a neutral pH.
Results and discussion
The uorescence spectroscopy of nanoprobe 1 to Cu2+
Synthesis and characterization of nanoprobe 1 3+
The Fe3O4 NPs and Fe3O4-DBA-PEG-NH-DTPA-AMC:Eu nanoprobe (1) were synthesized using a previous method.30,31 The uorescent nanoprobe consists of magnetic Fe3O4 NPs modied by polyethylene glycol-3,4-dihydroxybenzylamine (DBA-PEG-NH2) and Eu3+ ions integrated with a polyaminocarboxylate-based chelator (diethylene triamine pentaacetic acid DTPA). The organic chromophore, 7-amino-4-methyl coumarin (AMC) acted as an antenna absorbing excitation light and as a donor transferring energy to the Eu3+. According to potentiometric studies,32 the DTPA-AMC has a higher binding constant for Cu2+ (K ¼ 1021.77) than Eu3+ (K ¼ 1019.67, see ESI, Fig. S1, Tables S1 and S2†). Consequently, in the absence of Cu2+, this lanthanide–NPs complex emits the characteristic red uorescence of Eu3+. In the presence of Cu2+, Eu3+ can be replaced by Cu2+ to form Fe3O4-DBA-PEG-NH-DTPA-AMC:Cu2+ (2), which results in the uorescent quenching of 1. The transmission electron microscopy (TEM) images indicate that the morphology of as-synthesized Fe3O4 NPs and nanoprobe 1 were similar, and 1 could easily disperse in water (Fig. 1A and B). Effects of time and pH on the nanoprobe 1 To understand the quenching rate of Fe3O4-DBA-PEG-NHDTPA-AMC:Eu3+ by Cu2+, the uorescent intensity of 1 was
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The sensing ability of 1 for Cu2+ was investigated in an aqueous solution of Tris–HCl buffer (50 mM, pH 7.20) using uorescence emission spectroscopy. As shown in Fig. 2A, upon excitation at 325 nm, the solution of 1 showed the emission of europium at 580 nm, 595 nm and 616 nm via an intramolecular energy transfer from AMC to Eu3+. However, upon addition of Cu2+ at concentrations from 1 mM to 200 mM, the emission intensity of 1 gradually decreased, indicating that Eu3+ was replaced by Cu2+. Consequently, the emission color of the probe showed a dramatic change from red to colorless in the presence of Cu2+ (Fig. S4†). Also, the change between the uorescent intensities
Fig. 2 (A) Fluorescent emission spectra of 10 mM 1 upon addition of Cu2+ at concentrations from 1 mM to 200 mM in Tris–HCl buffer (50 mM, pH 7.20). (B) The linear relationship between the fluorescent intensity of 10 mM of 1 and Cu2+ concentrations from 0.1 nM to 1 nM at 616 nm.
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of 1 and the concentrations of Cu2+ in the range 0.1–1 nM shows a good linear correlation (Fig. 2B). The detection limit (DL) based on the formation of 2 is 6.35 ppt. This DL for Cu2+ is far less than DLs obtained with other probes based on organic uorophores13,33 and is sufficiently lower than the acceptable limit of the US EPA value of Cu2+ ions (1.3 ppm) in drinking water, indicating that the probe 1 is sensitive for detecting Cu2+ in aqueous solutions. Selectivity of nanoprobe 1 for Cu2+ To check whether nanoprobe 1 can selectively detect Cu2+, the effects of other metal ions on the uorescence of 1 have also been examined to evaluate the selectivity under the same experimental conditions. As shown in Fig. 3A (red bars), only the addition of Cu2+ resulted in a great uorescent intensity reduction from 117 a.u. to 30 a.u. at 616 nm, and the uorescent color of the probe changed from red to colorless. In contrast, the addition of large excesses of other metal ions, such as: Ag+, Al3+, Ba2+, Ca2+, Cd2+, Co2+, Cr3+, Fe2+, Fe3+, Hg2+, K+, Li+, Mg2+, Mn2+, Na+, Ni2+, Pb2+ and Zn2+, caused only slight changes, which were easily observed by the naked eye with irradiation from a UV-lamp (Fig. S5†). Therefore, nanoprobe 1 displayed high selectivity for the luminescent sensing of Cu2+. To further explore the use of nanoprobe 1 as an ion-selective nanosensor for Cu2+, competitive experiments were performed by adding Cu2+ to solutions of 1 in the presence of other cations. As shown in Fig. 3A (blue bars), signicant quenching of uorescence was observed for 1 upon the addition of Cu2+ in the presence of other metal ions. The results indicate that the sensing of Cu2+ by 1 is hardly disturbed by these commonly coexisting ions. Therefore, 1 can act as a highly selective probe for Cu2+. Quenching mechanism of Cu2+ In order to further understand the quenching mechanism of Cu2+ with 1, uorescence lifetime and the characteristic features of the uorescence intensity-based Stern–Volmer plot of 1 in the presence and absence of Cu2+ were investigated. As shown in Fig. S6 and Table S3,† the luminescent lifetimes of complexes 1 and 2 were measured by tting them to
Fig. 3 Effects of various metal ions on the fluorescent intensity of 10 mM of 1 in Tris–HCl buffer (50 mM, pH 7.20). The red bars represent the emission intensity of 1 in the presence of interfering ions (100 mM) at 616 nm. The blue bars represent the emission intensity of 1 upon addition of Cu2+ (100 mM) in the presence of each interfering metal ion (100 mM).
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biexponential decay curves. In the absence of Cu2+, biexponential behavior was observed with s values (relative weighting) of 0.56 ms (56%) and 1.16 ms (44%). In the presence of 7 mM Cu2+, the corresponding values were 0.41 ms (51%) and 1.06 ms (49%). Upon the addition of Cu2+, the average emission lifetime was reduced from 0.83 to 0.73 ms, indicating that excited-state molecules of 1 interacted with Cu2+. Fig. S7† shows the intensity-based Stern–Volmer plot of F0/F versus [Cu2+]. Interestingly, F0/F versus [Cu2+] plots show a distinct upwards curvature, implying that the 1–Cu2+ uorophore–quencher pair in solution does not show simple Stern–Volmer behavior20,34 and the static quenching and dynamic quenching occur concomitantly in this case. The static quenching is the predominant mechanism because the F0/F ¼ 1.51 is a higher value than hs0i/hsi ¼ 1.13 at 7 mM Cu2+. hs0i and hsi are average lifetimes of 1 in the absence and presence of Cu2+, respectively. Considering the following equations: 2 F0 ¼ 1 þ ðKD þ KS Þ Cu2þ þ KD KS Cu2þ F
(1)
F0 ¼ 1 þ KD Cu2þ 1 þ KS Cu2þ F
(2)
F0 ¼ 1 þ Kapp Cu2þ F
(3)
Kapp ¼
F0 1 1 ¼ ðKD þ KS Þ þ KD KS Cu2þ ½Cu2þ F KS2 KSI + S ¼ 0
(4)
(5)
The apparent quenching constant is calculated at each quenching concentration. A plot of Kapp versus [Cu2+] yields a straight line with an intercept of KD + KS and a slope of KDKS. The individual values can be obtained from the two solutions of the quadratic equation. The KD and KS were found to be 2.62 103 M1 and 7.65 103 M1, respectively. Separation of Cu2+ in water Magnetic separation is regarded as one of the most promising methods for Cu2+ removal from water. An extremely sensitive method based on uorescent spectroscopy was employed to detect free copper ions aer the magnetic separation, and then the separation efficiency was evaluated. Because 1 exhibits superparamagnetism at room temperature (Fig. S8†), we attempted to use 2 formed by adding Cu2+ to 1 for indirectly separating the Cu2+ from the aqueous solution. The different amounts of nanocomposite 1 (0, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4 mg) were added into 2 mL of a Cu2+ (4 ppm) aqueous solution, and then the mixture was gently stirred for 1 h at room temperature. Aer reaction, the hybrid formed, 2, containing Cu2+ was enriched and separated using a magnet, and the supernatant solution was collected for the further evaluation of the residual Cu2+ concentrations and the separation efficiency. The separation efficiency was dened as the decrease in the Cu2+ concentration before and aer magnetic separation. There was a quasiquantitative relationship between the Cu2+ concentration and
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Fig. 4 (A) The residual Cu2+ concentrations in aqueous solution after magnetic separation using different amounts of 1 (0, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4 mg). (B) Removal efficiency using different amounts of 1 for removal of Cu2+ in aqueous solution.
the uorescent intensity of nanoprobe 1. Firstly, the uorescent intensity of the standard compound AMC-DTPA-NH-PEGDBA:Eu3+ (105 M) with and without the addition of Cu2+ (4 ppm) standard solution was set as F0 and F, and DFS was dened as the value of the difference between F0 and F. Therefore, DFS acting as the reference standard was used to estimate the residual Cu2+ concentrations and the separation efficiency of the following procedures. Secondly, the Cu2+ concentration in the supernatant solution containing different amounts of nanoprobe 1 aer magnetic separation was indirectly detected by using the standard compound AMC-DTPA-NH-PEGDBA:Eu3+ (105 M), where the uorescent intensity changes were set as DFx. Finally, the results from the previous two steps were compared to estimate the approximate Cu2+ concentration and to calculate the separation efficiency. In addition, every Cu2+ concentration in the supernatant solution was prepared from a Cu2+ standard concentration and again veried by using the standard compound AMC-DTPA-NH-PEG-DBA:Eu3+ (Fig. S9 and Table S4†). As shown in Fig. 4A, when the dispersion amount of 1 in water reaches 1 mg, the concentration of Cu2+ decreases to 1.18 ppm, which is less than the US EPA drinking water standard (1.3 ppm). The investigation of Cu2+ separation efficiency was carried out as shown in Fig. 4B. By comparing the Cu2+ concentration before and aer the magnetic separation, more than 70% of Cu2+ was removed during the 1 h collection which corresponded to 1 mg of nanocomposite 1. The Eu3+ ions exchanged in solution could be detected and were removed by the Fe3O4-DBA-PEG-NH-DTPA-AMC (Fig. S10†). These experimental results demonstrated that 1 could not only be used as a detection reagent for Cu2+, but could also be used as a water purication agent to remove any excess copper ions.
Fluorescence imaging The ability of nanocomposite 1 to detect Cu2+ within living cells was also evaluated by using laser confocal uorescence imaging. Under selective excitation at 325 nm, HeLa cells incubated with nanocomposite 1 (10 mM Eu3+) for 1 h at 37 C showed the characteristic red emission uorescence of europium (Fig. 5A). When the nanocomposite 1-loaded cells were incubated with 35 mM Cu2+ for another hour, the red uorescence was almost quenched (Fig. 5B). This implies that
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Fluorescent images of HeLa cells (A) after incubation with 1 for 1 h and (B) after addition of 35 mM Cu2+ to the 1 treated cells for 1 h. Excitation at 325 nm and emission at 616 nm. Fig. 5
nanocomposite 1 could be used to monitor Cu2+ in living cells.
Conclusion In conclusion, lanthanide-functionalized magnetic nanoparticles were prepared that act as a new type of uorescent sensor for ultrasensitive sensing and efficiently separating Cu2+ in aqueous solution. These multi-functional nanoparticles exhibit a high sensitivity and selectivity for targeting Cu2+ ions over various other metal ions, with a detection limit of 0.1 nM. Aer interaction with Cu2+, the characteristic red uorescence of europium was quenched and the uorescent color changed to colorless. The sensor also exhibited high selectivity for Cu2+ over other common metal ions. Moreover, 1 was shown to be stable in aqueous solution over a wide pH range and was suitable for uorescent imaging in living cells, which indicates that 1 has potential applications in biological analysis. Furthermore, copper ions could be collected and separated using these magnetic nanoprobes with a commercial magnet. The experimental results reported here open up a new method for simple and reliable monitoring of environmentally toxic ions as well as enrichment/removal of excess toxic ions.
Experimental Chemicals Iron(III) acetylacetonate, oleylamine, benzyl ether, 3,4-dihydroxybenzaldehyde, 7-amino-4-methyl coumarin (AMC), and PEG (MW ¼ 4000) were purchased from Sigma-Aldrich and were used without further purication. Diethylenetriaminepentaacetic acid dianhydride (DTPAA),35 1,u-diaminopolyoxyethylene (MW ¼ 4000),36 polyethylene glycol-3,4-dihydroxybenzylamine (H2N-PEG-DBA, MW ¼ 4000),27 Fe3O4 nanoparticles,30 and Fe3O4-DBA-PEG-NH-DTPA-AMC:Eu3+ (1)31 were synthesized according to the published method. The solution of metal ions (AgNO3, Al(NO3)3, BaCl2, Ca(NO3)2, Cd(NO3)2, Co(NO3)2, CrCl3, CuCl2, FeCl3, FeSO4, Hg(ClO4)2, KNO3, LiBr, Mg(NO3)2, Mn(CH3COO)2, NaNO3, Ni(NO3)2, Pb(CH3COO)2, Zn(NO3)2) were dissolved in ultrapure water. Aqueous Tris–HCl buffer (50 mM, pH 7.20, NaCl 50 mM) was used to maintain the pH value and the ionic strength of all the solutions used in the
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experiments. EuCl3$6H2O was obtained by dissolving Eu2O3 in hydrochloric acid followed by successive fuming to remove excess acid.
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Instrumentation The uorescence spectra were recorded on a Shimadzu RF-5301 spectrouorophotometer. The uorescence lifetimes were recorded on the Edinburgh Photonics FLS920 steady statetransient state uorescence spectrometer. The TEM measurements were carried out using a Philips EM 420 (120 kV). Fluorescent images were taken on Zeiss Leica inverted epiuorescence/reectance laser scanning confocal microscope. The concentrations of Eu3+ were determined by inductively-coupled plasma – atomic emission spectroscopy (ICPAES). The hysteresis loop was obtained at 300 K with a Lake Shore Cryotronics 7400 VSM system. Preparation of Fe3O4-DBA-PEG-NH-DTPA-AMC:Eu3+ (1) Fe3O4-DBA-PEG-NH-DTPA-AMC:Eu3+ (1) was synthesized according to the previously published method. Briey, 35.7 mg of DTPAA (0.1 mmol) and 17.5 mg AMC (0.1 mmol) were dissolved in 2 mL and 1 mL of anhydrous dimethylformamide (DMF), respectively. Dry triethylamine (100 mL) was added to the DTPAA solution. Then, the DMF solutions of DTPAA and AMC were mixed, and the mixture was stirred for 10 h at room temperature. Then 10 mL of a dry CH2Cl2 solution of 0.4 g DBAPEG-NH2 (0.1 mmol) was added to the DMF solution of DTPAAMC, and the mixture obtained was stirred for 24 h at room temperature. Then EuCl3$6H2O 36.7 mg (0.1 mmol) was added at an equivalent molar rate to the AMC-DTPA-NH-PEG-DBA solution for 12 h with stirring at room temperature. The product was precipitated by adding diethyl ether and collected by centrifugation. Aer washing with DMF, CH2Cl2 and diethyl ether, the product Eu3+:AMC-DTPA-NH-PEG-DBA was then dried under vacuum. The CHCl3 solution of Fe3O4 nanoparticles was added to 100 mg of the Eu(III) complex DMF solution and then stirred for 12 h at room temperature. The product was precipitated by adding petroleum ether, collected by centrifugation at 4000 rpm, washed with DMF, ethanol and petroleum ether, and then redispersed in water and dialysed with H2O for 24 h to remove unreacted organic molecules. 32
Potentiometric studies
The protonation constant of the ligand (DTPA-AMC) and the stability constant (i.e., binding constant) of the complexes (Eu:DTPA-AMC and Cu:DTPA-AMC) were studied at 25 C and at an ionic strength of I ¼ 0.10 mol dm3 (NaCl) using potentiometric pH titration. The protonation constant of the ligand was calculated rst and this was then used for determining the stability constant of the complexes. Potentiometric titrations were carried out using the ligand DTPA-AMC for calculating the protonation constants of the ligand. Then, the ligand DTPAAMC and two different metal solutions (Eu3+ and Cu2+) were used for calculating the stability constant of the metal complexes. Each of the potentiometric measurements were performed by titrating 50 mL of the aqueous NaCl solutions
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against the standard NaOH solutions until the formation complexes were stable. The overall experimental procedure involved the potentiometric titrations of the following solutions: (a) 10 mL of 1 mM ligands + 50 mL of 0.1 M NaCl (for the determination of the protonation constant of ligand), (b) 10 mL of 1 mM ligands + 10 mL of 1 mM metal ion + 50 mL of 0.1 M NaCl (for the determination of the stability constant of complexes). The computations of protonation constant of ligand and the stability constant of complexes from potentiometric data were carried out using the Bjerrun's Half-n Method. All of the potentiometric data were recorded between pH 3.0 and 10.0. Fluorescence spectral studies Stock solutions of various metal ions were prepared in ultrapure water. A stock solution of 1 (0.5 mM Eu3+) was analysed by ICPAES in pure water. The solution of 1 was then diluted to 10 mM with Tris–HCl (50 mM, pH 7.20). In titration experiments, a 2 mL solution of 1 (10 mM) was placed in to a quartz optical cell with a 1 cm optical path length, and the Cu2+ solutions from 1 mM to 200 mM, were gradually added into the quartz optical cell using a micropipette. In selectivity experiments, the test samples were prepared by using appropriate amounts of the metal ion solution. Finally, the concentration of solution 1 was 10 mM and the metal ion concentration was 100 mM. To study the coexistence of Cu2+ and other metal ions studies, 100 mM Cu2+ was then added to the solutions of 1 and metal ions. In reaction time experiments, the uorescent intensity of 10 mM 1 was measured at different time intervals in the presence of 1 mM, 10 mM and 100 mM Cu2+ in an aqueous solution of Tris–HCl. In pH effect experiments, the uorescent intensity of 10 mM of 1 was measured without and with 10 mM Cu2+ at different pH values. The pH values were regulated in the range of 3 to 11 using diluted hydrochloric acid or sodium hydroxide. For uorescence measurements, excitation was at 325 nm, and emission was from 560 to 640 nm. There are three special peaks at 580 nm, 595 nm, 616 nm and the emission intensity at 616 nm was recorded in some experiments. Removal of Cu2+ by Fe3O4-DBA-PEG-NH-DTPA-AMC:Eu3+ (1) Preparation of standard samples. The Eu3+:AMC-DTPA-NHPEG-DBA standard solution were prepared to give a concentration of 10 mM Eu3+. Aer addition of CuCl2 solution (4 mg L1), the uorescence intensity was measured. Different amounts of 1 from 0, 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4 mg were dispersed into 2 m of CuCl2 solution (4 mg L1). Aer stirring the mixture for about 1 h, the dispersed substance was separated using an external magnetic eld and centrifugation for separation of the mixture using precipitation, and then the residual concentrations of Cu2+ in water were measured by comparison with a 10 mM Eu3+:AMC-DTPA-NH-PEG-DBA standard solution. Lifetime The lifetime decay curves were recorded on the FLS920 steady state-transient state uorescence spectrometer. Excitation was Nanoscale, 2014, 6, 11473–11478 | 11477
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at 325 nm, and emission was at 616 nm. The lifetimes (s) of 1 (50 mM, s0) and 7 mM Cu2+ reactions with 1 (50 mM) formed nanocomposite 2 (s) were measured, respectively. The average lifetimes hs0i (1), hsi (2) were calculated using the equation hsi ¼ P 2P Aisi / Aisi.37
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Fluorescence microscopic imaging HeLa cells were grown in Dulbecco's modied Eagle medium supplemented with 10% fetal bovine serum in an atmosphere of 5% CO2 and 95% humidied air at 37 C for 24 h. Then the cells were treated with the various compounds using uorescence microscopic imaging. The cultured cells were treated with 1 and incubated for 1 h. Subsequently, the cells were treated with 35 mM CuCl2 for another 1 h. Aer incubation for the corresponding time, the cells were washed three times with phosphate buffered saline to remove free compounds and ions before analysis. Then uorescence microscopic images were acquired. All confocal images were collected with a Zeiss Leica inverted epiuorescence/reectance laser scanning confocal microscope with excitation at 325 nm and emission at 616 nm.
Acknowledgements The work was supported by the National Natural Science Foundation of China (21271093), the NCET (13-0262), and the Fundamental Research Funds for the Central Universities (lzujbky-2013-56 and lzujbky-2014-k06).
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