Biosensors and Bioelectronics 63 (2015) 566–571
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Lanthanide based dual-emission fluorescent probe for detection of mercury (II) in milk Hongliang Tan n, Qian Li, Chanjiao Ma, Yonghai Song, Fugang Xu, Shouhui Chen, Li Wang n Key Laboratory of Functional Small Organic Molecule, Ministry of Education, Key Laboratory of Chemical Biology of Jiangxi Province, College of Chemistry and Chemical Engineering, Jiangxi Normal University, Nanchang, 330022, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 16 April 2014 Received in revised form 21 July 2014 Accepted 11 August 2014 Available online 17 August 2014
It is highly desirable to develop a simple and sensitive method for Hg2 þ detection because of the dangerous nature of Hg2 þ . In this work, we prepared a dual-emission fluorescent probe for Hg2 þ detection by combining two lanthanide chelates with different emission wavelengths. Green-emitting terbium (Tb3 þ ) chelates as reference signals were embedded into SiO2 nanoparticles and red-emitting europium (Eu3 þ ) chelates as response units were covalently linked to the surface of silica shell. Upon the addition of Hg2 þ , the fluorescence of Eu3 þ chelates can be selectively quenched, while the fluorescence of Tb3 þ chelates remained unchanged. As a kind of Hg2 þ nanosensor, the dual-emission fluorescent probe exhibited excellent selectivity to Hg2 þ and high sensitivity up to 7.07 nM detection limit. The Hg2 þ levels in drinking water and milk samples were determined by using the dual-emission fluorescent probe with satisfied recovery. Additionally, our probe has a long enough fluorescence lifetime, which can avoid the interference from autofluorescence of the biological samples. We envision that the proposed probe could find great potential applications for ultrasensitive time-resolved fluorometric assays and biomedical imaging in the future. & 2014 Elsevier B.V. All rights reserved.
Keywords: Lanthanide Dual-emission Fluorescent probe Mercury (II) Milk
1. Introduction Mercury is one of the most toxic elements in ecosystems. Contamination of the environment with mercury is an important concern throughout the world (Clarkson, 1997). It is estimated that the total global mercury emissions from all sources – both natural and human-generated – reached nearly 7500 ton per year (EPA, 2005). Water soluble divalent mercuric ion (Hg2 þ ) is one of the most usual and stable forms of mercury pollution. Because of its non-biodegradation, Hg2 þ cannot be destroyed once it was released into the environment. Hg2 þ can be absorbed by crops, then contaminate food products, such as vegetables, rice, fruit, and even dairy products (Akkemik et al., 2012). The subsequent accumulation of Hg2 þ by humans through food chain can cause the damage of brain, kidneys, nervous system, and endocrine system (Nolan and Lippard, 2008; Zalups, 2000). Therefore, monitoring of the Hg2 þ level in environmental and biological samples is an important issue to understand its distribution and potential pollution.
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Corresponding authors. Tel./fax: þ86 791 88120861. E-mail addresses: [email protected] (H. Tan), [email protected] (L. Wang). http://dx.doi.org/10.1016/j.bios.2014.08.015 0956-5663/& 2014 Elsevier B.V. All rights reserved.
To date, many methods have been developed for the detection of Hg2 þ , such as atomic absorption spectrometry (Labatzke and Schlemmer, 2004), inductively coupled plasma mass spectrometry (Wang et al., 2007), electrochemistry (Mahajan et al., 2003) and so on. These analytical methods can measure Hg2 þ levels sensitively and accurately, but they require expensive and sophisticated equipment and/or involve tedious sample preparation procedures prior to the analysis, which limits their applications in laboratory. Fluorescent detection of Hg2 þ would be a more desirable method due to its operational simplicity, easy preparation of sample and high sensitivity. Over the past few years, some fluorescent probes based on organic molecules (Wang et al., 2006; Yoon et al., 2007; Zhu et al., 2006), conjugated polymers (Liu et al., 2007; Zhao and Zhong, 2006), quantum dots (Chen et al., 2006; Duan et al., 2009) and carbon dots (Lu et al., 2012; Zhou et al., 2012) have been applied to detect Hg2 þ . Particularly, lanthanide-based fluorescent probes for Hg2 þ detection have been paid considerable attention due to their unique optical properties including large Storks shift, sharp emission, high quantum yields and long lifetime. These properties enable the lanthanide-based fluorescent probes to eliminate efficiently the interferences from background and scattering fluorescence (Richardson, 1982). Nevertheless, few studies have centered on using lanthanide-based fluorescent probes for the detection of Hg2 þ .
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In the previous works, we reported two different methods to detect Hg2 þ using terbium (Tb3 þ ) chelate (Tan et al., 2011) and lanthanide coordination polymers (Tan et al., 2012) as fluorescent probes, respectively. The former was designed based on the fluorescence quenching of Tb3 þ chelate which resulted from the replacement of Tb3 þ by Hg2 þ , and the latter was constructed on the basis of Hg2 þ -induced fluorescence enhancement through a process of photoinduced electron transfer. Although these two lanthanide fluorescent probes exhibited high sensitivity and excellent selectivity for Hg2 þ , they were single wavelength intensity-based detection methods and tended to be affected by the concentration change of probes and/or the microenvironment of complex samples. Recently, dual-emission fluorescent probebased detection methods have received considerable attention. Compared with single wavelength intensity-based detection methods, the signal variation of dual-emission fluorescent probe is easier to be distinguished by naked eyes. Moreover, dualemission fluorescent probe may provide improved quantification accuracy because of its independence of probe concentration (Qu et al., 2013; Zhang et al., 2011). Dual-emission fluorescent probe is usually constructed by combining two fluorophores with different emission wavelengths, one fluorophore functions as reference unit and another as response moiety. Up to now, many fluorophores including organic dyes (Yilmaz et al., 2010; Zong et al., 2011), quantum dots (Zhang et al., 2011), carbon dots (Liu et al., 2014; Zhu et al., 2012), and gold cluster (Wang et al., 2014) have been employed to construct dual-emission fluorescent probes. In spite of this, the development of dual-emission fluorescent probe for Hg2 þ detection is still limited by the design of new organic fluorophores which are specific and sensitive to Hg2 þ . In this work, we attempt to employ two lanthanide chelates with different emission wavelengths to prepare a dual-emission fluorescent probe for the detection of Hg2 þ . Two lanthanide chelates were synthesized by the chemical coordination of terbium ion (Tb3 þ ) and europium ion (Eu3 þ ) with dipicolinic acid (DPA, 2.6-pyridinedicarboxylic acid), respectively (denoted as TbDPA chelate and Eu-DPA chelate). Owing to the sensitization effect of DPA, Tb-DPA chelate emits strong green fluorescence, whereas a strong red fluorescence can be observed from Eu-DPA chelate. To construct a dual-emission fluorescent probe (Tb-DPA@SiO2-EuDPA), Tb-DPA chelate was embedded into SiO2 nanoparticle as reference signal and Eu-DPA chelate was immobilized on the
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surface of silica shell as response unit. Apart from the sensitization effect, DPA is a very useful masking agent to eliminate the interference from other metal ions in various Hg2 þ sensor systems (Chen et al., 2011; Darbha et al., 2007; Huang and Chang, 2006; Liu et al., 2012). Thus, Tb-DPA@SiO2-Eu-DPA might possess the potential as a dual-emission fluorescent probe for selective detection of Hg2 þ . In addition, the surface of SiO2 nanoparticle dropped with Tb-DPA chelate was functionalized by diethylenetriaminepentaacetic acid (DTPA) to immobilize Eu-DPA chelate. DTPA was selected as the functional ligand; on the one hand, it can offer its carboxyl groups to coordinate with Eu-DPA chelate, and leading to the formation of DTPA-Eu-DPA ternary complex on surface of the SiO2 nanoparticle. On the other hand, DTPA might assist DPA to selective response to Hg2 þ due to its higher binding constant to Hg2 þ (K ¼1026.4) than Eu3 þ (K ¼1022.39) (Holloway and Reilley, 1960; Patnaik and Dean, 2004). In the presence of Hg2 þ , therefore, quenched Eu3 þ fluorescence on the surface of SiO2 nanoparticle would be observed due to the replacement of Eu3 þ by Hg2 þ (Scheme 1).
2. Material and methods 2.1. Chemicals and solutions Terbium nitrate (99.99%) and europium nitrate (99.99%) were purchased from Ruike Rare Earth Metallurgy and Functional Materials Co., Ltd. (Baotou, China); Dipicolinic acid (99%, 2, 6-pyridinedicarboxylic acid) and diethylenetriaminepentaacetic acid dianhydride (98%, DTPA dianhydride) was purchased from Sigma-Aldrich (Shanghai, China); Tetraethyl orthosilicate (99%, TEOS); 1-hexanol (99%); cyclohexane (99.5%), and metal salts (Hg(NO3)2, AgNO3, MgCl2, Pb(NO3)2, Zn(NO3)2, CdCl2, FeCl3, FeCl2, NiCl2, CoCl2, MnCl2, CrCl3 and CuCl2) were purchased from Sinopharm Chemical Reagent Company (Shanghai, China). 3-aminopropyltriethoxysilane (99%, APTES), N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) were obtained from Aladdin (Shanghai, China). Ultrapure water (18 MΩ cm; Milli-Q, Millipore) was used for the preparation of all aqueous solutions. Unless otherwise stated, all chemicals were of analytical reagent grade and were used without further purification.
Scheme 1. . Illustration of dual-emission fluorescent probe Tb-DPA@SiO2-Eu-DPA for the detection of Hg2 þ .
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2.2. Instruments The morphology of the as-prepared SiO2 nanoparticles was observed by transmission electron microscopy (TEM, JEM-2100, Japan). Fluorescence spectra and emission intensity were recorded on an LS55 luminescence spectrometer (PerkinElmer, UK). The detection solution was placed in a quartz micro-cuvette with 100 μL capacity and 2 mm lightpath. The 270-nm excitation wavelength was used for the emission spectra. A delay time of 0.05 ms and a gate time of 2 ms were used. For the emission lifetime, the fluorescence intensities at 545 nm for Tb3 þ and 615 nm for Eu3 þ were recorded under different delay times and fitted with an exponential function. UV–visible absorption spectra were recorded with a UV-3900H spectrophotometer (Hitachi, Japan). All the experiments were performed at room temperature. All error bars represent standard deviations from three repeated experiments. 2.3. Synthesis of Tb-DPA-APTES conjugate To covalently link Tb-DPA chelate to the silica matrix, Tb-DPAAPTES conjugate was synthesized. Briefly, 600 μL of 20 mM DPA aqueous solution was added to 500 μL anhydrous ethanol solution containing 9.0 mg of EDC and 2.8 mg of NHS and stirred for 30 min. Then, 100 μL of APTES was added to the mixture. After reacting for 2 h, 200 μL of 20 mM Tb(NO3)3 aqueous solutions was added under stirring. The reaction was kept for another 15 min. The obtained solution containing Tb-DPA-APTES conjugate was used for preparing SiO2 nanoparticles without further manipulation. 2.4. Preparation of amino-modified Tb-DPA doped SiO2 nanoparticles The Tb-DPA doped SiO2 nanoparticles (Tb-DPA@SiO2) were prepared in a water-in-oil (W/O) reverse microemulsion with the following procedure (Chen et al., 2007). To a microemulsion solution containing 4 mL of cyclohexane, 1 mL of n-hexanol, and 1 mL of Triton X-100 were added to 295 μL of Tb-DPA-APTES conjugate under stirring. After reacting for 30 min at room temperature, 85 μL of TEOS and 25 μL of ammonia solution (28%) were added. The reaction was continued for 24 h. The nanoparticles were isolated from the microemulsion by adding equal volume of acetone, followed by centrifuging and washing with ethanol and water several times to remove the surfactant and unreacted materials. To functionalize the as-prepared
nanoparticles with amino group, the cleaned nanoparticles were firstly dispersed in the microemulsion solution with the same components. Then, 20 μL of APTES was added to the mixture. After the reaction was carried out for 2 h, the nanoparticles were collected and washed by the same steps as above. 2.5. Modification of Tb-DPA@SiO2 with Eu-DPA chelate A quantity of 2 mg of NH2-modified Tb-DPA@SiO2 was suspended in 3 mL of PBS (phosphate buffered saline, 0.01 M, pH 7.4) and 20 mg of DTPA dianhydride was then added to this solution. After stirring for 2 h, the resulting mixture was collected and washed by centrifuging and ultrasonic dispersion. The washed nanoparticles were then dispersed in 3 mL of ultrapure water. And 300 μL of Eu-DPA chelate obtained by mixing Eu(NO3)3 and DPA aqueous solution with a molar ratio of 1:3 was added. The reaction was incubated for 30 min at room temperature. Those unbonded Eu-DPA chelates were removed by centrifuging. Finally, the precipitate was dispersed in 3 mL of ultrapure water to form Tb-DPA@SiO2-Eu-DPA suspension and stored 4 °C for further use. 2.6. Detection of Hg2 þ in aqueous solution For Hg2 þ detection, solutions with Hg2 þ concentrations from 0 to 10 μM were added into 10 μL of Tb-DPA@SiO2-Eu-DPA suspension, and H2O was added till the total volume reached to 100 μL. After reacting for 10 min, the fluorescence spectra were recorded using an excitation wavelength of 270 nm. To test the selectivity of Tb-DPA@SiO2-Eu-DPA to Hg2 þ , 6 μL of 10 mM stock solutions of interference metal ions was added into 10 μL of Tb-DPA@SiO2-Eu-DPA suspension, respectively. The total volume was reached to 100 μL by adding ultrapure water. The reaction was lasted 10 min before measuring the fluorescence intensities of these mixtures at 615 nm. 2.7. Detection of Hg2 þ in drinking water and milk The drinking water and milk samples were obtained from a local supermarket. To detect Hg2 þ in drinking water, different volumes of standard solutions of Hg2 þ were spiked into drinking water to prepare water samples containing different concentrations of Hg2 þ . Then, 10 μL of Tb-DPA@SiO2-Eu-DPA suspension was added to these water samples, respectively. After reacting for 10 min at room temperature, the fluorescence intensities at 615 nm of these mixtures were recorded under a 270-nm excitation wavelength. For the detection of Hg2 þ in milk, milk powders
Fig. 1. TEM images of Tb-DPA@SiO2-Eu-DPA SiO2 nanoparticles.
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were treated in following steps. Firstly, the proteins in milk powders were removed by adding 1% (v/v) trichloroacetic acid and sonicating for 20 min. Then the lipids were removed by collecting the supernatant filtered through a 0.22 mm membrane (Whatman) after centrifuging the sample at 12,000 rpm (14,463g) for 10 min. A series of milk samples containing different concentrations of Hg2 þ in the range of 0–1 μM was prepared by “spiking” them with different volumes of a stock solution of Hg2 þ (100 μM). Tb-DPA@SiO2-Eu-DPA suspension was then added to these milk samples, respectively; the total volume is 100 μL as mentioned above. The resulting solutions were mixed well and stood for 10 min before recording their emission spectra.
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between Tb-DPA chelate and Eu-DPA chelate was not occurred. Furthermore, it can be seen from Fig. 2 that the fluorescence of EuDPA chelate of the dual-emission probe showed a significant decrease upon the addition of Hg2 þ . By contrast, no marked changes (o5%) were found in the fluorescence of Tb-DPA chelate of the probe in the presence of Hg2 þ . The results suggest that the added Hg2 þ only quench the fluorescence of Eu-DPA chelates on the surface of silica shell and has no influences on the fluorescence of Tb-DPA chelate embedded into the silica core. Therefore, Tb-DPA@SiO2-Eu-DPA appears to be a useful dual-emission fluorescent probe for Hg2 þ detection. 3.2. Lifetime and absorption spectra
3. Results and discussion 3.1. Characterizations As shown in Fig. 1, the resultant SiO2 nanoparticles are uniform and monodispersed and have average sizes of 60 75 nm. The surface of the nanoparticle is rough, which may result from the conjugation of Eu-DPA chelates to the DTPA on the surface of silica shell. Moreover, a silica shell with thickness of approximately 10 nm can be observed. The existence of the silica shell is favorable to avoid the occurrence of fluorescence resonance energy transfer (FRET) process from Tb-DPA chelate to Eu-DPA chelate and to prevent the direct contact of the Tb-DPA chelate in the interior of SiO2 nanoparticles with the external solvents. Fig. 2 showed the fluorescent emission spectra of Eu3 þ functionalized Tb-DPA@SiO2 nanoparticles (Tb-DPA@SiO2-Eu3 þ ) under different conditions. In the absence of DPA, Tb-DPA@SiO2-Eu3 þ exhibited a typical emission of Tb3 þ with peaks at 490, 545, 584 and 620 nm. In the presence of DPA, however, two new emission peaks at 590 and 615 nm were observed except the existed emission peaks of Tb3 þ at 490 and 545 nm. The two new peaks can be assigned to 5D0 to 7 F1 and 5D0 to 7F2 electronic transitions of Eu3 þ , respectively (Richardson, 1982). The appearance of the emission peaks of Eu3 þ implies the occurrence of the chemical coordination of the added DPA with Eu3 þ on the surface of Tb-DPA chelates-doped SiO2 nanoparticles, which results in the formation of the dual-emission fluorescent probe Tb-DPA@SiO2-Eu-DPA. It was also noteworthy that no obvious changes in the fluorescence of Tb-DPA chelate as reference signals were observed after the formation of Eu-DPA on the surface of silica shell. This indicates that the FRET process
To understand the interaction of Hg2 þ with the dual-emission fluorescent probe, we investigated the changes of the emission lifetime of Tb-DPA@SiO2-Eu-DPA in the absence and presence of Hg2 þ . After the addition of Hg2 þ , the emission lifetime of Eu-DPA chelate on the surface of silica shell was decreased from its original 1.169 to 0.788 ms (Fig. S1). However, the emission lifetime of Tb-DPA chelate remained unchanged (data not shown). The results confirm that Hg2 þ only reacts with Eu-DPA chelate and displays no effects on the inherent transition processes of Tb3 þ induced by the ligand-field. In addition, the effects of Hg2 þ on the absorption spectra of the Tb-DPA@SiO2-Eu-DPA were examined. As shown in Fig. S2, Tb-DPA@SiO2-Eu-DPA alone exhibited an obvious absorption peak at 272 nm, which could be assigned to the maximum absorption of DPA molecules. Upon the addition of Hg2 þ , no marked changes in the absorption spectra of Tb-DPA@SiO2-Eu-DPA were observed except a slight change in the absorption intensity. Even when Hg2 þ concentration was increased to 1 mM, the absorption spectrum of Tb-DPA@SiO2-EuDPA was still not changed. The unchanged absorption spectra reflect that Eu3 þ co-coordinated to DTPA and DPA was replaced by Hg2 þ , which led to the formation of DTPA-Hg-DPA ternary complex on the surface of the silica shell. 3.3. Reaction time of Hg2 þ In order to determine the response rate of fluorescent signal upon the addition of Hg2 þ , time-dependent measurements on the fluorescence of Tb-DPA@SiO2-Eu-DPA to Hg2 þ with different concentrations were conducted. As shown in Fig. S3, with the addition of Hg2 þ , the fluorescent intensity of Tb-DPA@SiO2-EuDPA at 615 nm was rapidly decreased, and reached a steady value within 5 min. The decrease of the fluorescence intensity of Tb-DPA@SiO2-Eu-DPA at 615 nm is proportional to the concentration of Hg2 þ . This indicates that the quenching reaction of Hg2 þ to Tb-DPA@SiO2-Eu-DPA needs 5 min to complete and the decrease in the fluorescent intensity of Tb-DPA@SiO2-Eu-DPA at 615 nm is time-dependent. Thus, 5 min was chosen as the reaction time in the subsequent experiments to obtain the best sensitivity. 3.4. Detection sensitivity for Hg2 þ
Fig. 2. Emission spectra of Tb-DPA@SiO2-Eu3 þ (a), Tb-DPA@SiO2-Eu3 þ in the presence of DPA (b) and Tb-DPA@SiO2-Eu3 þ with the addition of DPA and 0.5 μM of Hg2 þ (c). The emission spectra were recorded at 270 nm excitation wavelength.
To evaluate the detection sensitivity of Tb-DPA@SiO2-Eu-DPA as a dual-emission fluorescent probe for Hg2 þ , the fluorescent responses of the probe to Hg2 þ with different concentrations were investigated. As shown in Fig. 3a, the fluorescence intensity of the probe at 615 nm was highly sensitive to Hg2 þ and decreased with the increase of Hg2 þ concentration. Nevertheless, the fluorescent intensity of the probe at 545 nm showed almost no changes. There is a good linear correlation between the intensity ratio of F545/F615 and the concentration of Hg2 þ in the range of 10 nM–2 μM (Fig. 3b). The detection limit is calculated to be 7.07 nM, which is lower than the maximum level (10 nM) of
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a
b
Fig. 4. The detection selectivity of Tb-DPA@SiO2-Eu-DPA in the presence of 6 μM various metal ions. Black bars represent the addition of single metal ion (6 μM); Red bars represent the addition of single metal ion (6 μM) and Hg2 þ (6 μM) together except Fe3 þ . The gray and red bars of Fe3 þ represent the addition of mixture of Fe3 þ and H2O2 in the absence and presence of Hg2 þ , respective. Relative standard deviations (RSD) are all less than 7.41%. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
displays excellent fluorescence stability at room temperature. No obvious changes in the fluorescent response of the dual-emission probe to Hg2 þ were observed after the coordination polymers were stored for 30 days (Fig. S4). This suggests that the dualemission probe possesses great potential as a fluorescence material to detect Hg2 þ . 3.5. Detection selectivity for Hg2 þ
c
Fig. 3. (a) Fluorescence emission spectra of Tb-DPA@SiO2-Eu-DPA in the presence of different concentrations of Hg2 þ solution. (b) The plot of F545/F615 as a function of the Hg2 þ concentration. Relative standard deviations (RSD) are all less than 4.39%. (c) Visual fluorescence color changes of the dual-emission fluorescent probe upon the addition of different concentrations of Hg2 þ (from left to right: 0, 0.04, 0.2, 1, 6 μM). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Hg2 þ in drinking water permitted by the U.S. Environmental Protection Agency (EPA) (EPA, 1988). Compared with other fluorescent methods for Hg2 þ detection (Chen et al., 2011; Duan et al., 2009, 2014; Mandal et al., 2013; Yan et al., 2013; Zhou et al., 2012), the presented dual-emission probe displayed a comparable detection limit. Additionally, Hg2 þ -induced fluorescence changes can be observed under a UV lamp. From Fig. 3c, it can be seen that the color of the dual-emission probe solution gradually changed from orange to green upon the titration with Hg2 þ , which enabled the naked-eye to detect Hg2 þ . Besides, the dual-emission probe
To evaluate the selectivity of the dual-emission probe for Hg2 þ , we investigated the response of the probe to other metal ions that often coexisted with Hg2 þ in the environment. These metal ions employed in our selective experiments included Ca2 þ , Cd2 þ , Co2 þ , Cr3 þ , Cu2 þ , Fe2 þ , Fe3 þ , K þ , Na þ , Mg2 þ , Mn2 þ , Ni2 þ and Pb2 þ . From Fig. 4, it can be found that Hg2 þ was the only metal ion which caused a significant change in F545/F615 of Tb-DPA@SiO2-EuDPA except Fe3 þ , and no obvious fluorescent responses can be found in the presence of the interfering metal ions. The interference of Fe3 þ can be eliminated by the addition of H2O2 because H2O2 can reduce Fe3 þ to Fe2 þ . Since the fluorescence of Tb-DPA@SiO2-Eu-DPA stays constant in the presence of H2O2 with different concentrations (data not shown), H2O2 has no effects on the detection of Hg2 þ . The selective response of Tb-DPA@SiO2-EuDPA to Hg2 þ can be attributed to the higher binding ability of DTPA to Hg2 þ than other interfering metal ions (Holloway and Reilley, 1960; Patnaik and Dean, 2004) and the selective recognition ability of DPA to Hg2 þ (Darbha et al., 2007; Huang and Chang, 2006). In addition, the potential effects of the interfering metal ions on the detection of Hg2 þ were assessed by examining the fluorescence changes of Tb-DPA@SiO2-Eu-DPA in the presence of 6 μM Hg2 þ and interferential metal ions (each 6 μM), respectively. The results were displayed in Fig. 4 (red bars), which indicates that the coexistence of these interfering metal ions has virtually no influence on the detection of Hg2 þ and further demonstrated the high specificity of the dual-emission probe for Hg2 þ . Due to the fact that milk might contain some substances that would affect the fluorescence of Eu-DPA chelate on the surface of silica shell by coordinating with Eu3 þ , such as amino acids, glucose and vitamins, the fluorescent responses of the probe to these substances were then studied. As shown in Fig. S5, no remarkable fluorescence responses were observed upon the
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addition of these amino acids (Arg, Tyr, Asp, Glu, His, Lys, Cys), ascorbic acid, fructose, lactose, glucose, Vit B1 and Vit B12; only Hg2 þ can cause the decrease of the fluorescence of Tb-DPA@SiO2 -Eu-DPA at 615 nm. The results suggest that the influences from these foreign substances are negligible, and the presented dualemission probe can be used for the detection of Hg2 þ in milk sample with high selectivity.
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Scientist Foundation of Jiangxi Province (20112BCB23006 and 20122BCB23011).
Appendix A. Supplementary information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.08.015.
3.6. Detection of Hg2 þ in drinking water and milk samples The presented dual-emission probe was further employed to determinate the levels of Hg2 þ in drinking water and milk samples. To detect Hg2 þ in drinking water and milk samples, standard addition method was used. As shown in Table S1, the recoveries of Hg2 þ in drinking water were between 97.2% and 105.4%, while for milk samples they were between 97.05% and 114.82%. The relative standard deviations (RSD, n ¼3) are all less than 2.35%. The results indicate that the dual-emission probe has good precision and satisfactory reproducibility. It is noteworthy that our analysis works admirably in highly low matrix samples that are not representative of real contaminated food.
4. Conclusion In summary, we have prepared a lanthanide-based dual-emission fluorescent probe for Hg2 þ detection by employing Tb-DPA and Eu-DPA chelates as reference signal and response moiety, respectively. The dual-emission fluorescent probe exhibited excellent selectivity to Hg2 þ and high sensitivity up to 7.07 nM detection limit. Compared with other dual-emission fluorescent methods, our fluorescent probe displayed a comparable detection limit and possessed a long enough fluorescence lifetime for the time-resolved fluorescence assays, which was especially advantageous for the detection of biosamples with autofluorescence. To the best of our knowledge, this is the first report on using two lanthanide chelates with different emission wavelengths to construct a dual-emission fluorescent probe. Furthermore, the resulted fluorescent probe was employed to detect the level of Hg2 þ in drinking water and milk samples and satisfactory results were obtained. We believe that the proposed strategy can be extended to the preparation of other lanthanide-based dual-emission fluorescent probes for the wide applications in biosensing, imaging, environmental analysis and so on.
Acknowledgment This work was supported by National Natural Science Foundation of China (Nos. 21305054 and 21165010), Scientific Research Foundation of Jiangxi Normal University, Specialized Research Fund for the Doctoral Program of Higher Education of China (No. 20133604120002), Scientific Research Foundation of Education Commission of Jiangxi Province (No. GJJ14258), and Young
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Lanthanide based dual-emission fluorescent probe for detection of mercury (II) in milk Hongliang Tan n, Qian Li, Chanjiao Ma, Yonghai Song, Fugang Xu, Shouhui Chen, Li Wang n Key Laboratory of Functional Small Organic Molecule, Ministry of Education, Key Laboratory of Chemical Biology of Jiangxi Province, College of Chemistry and Chemical Engineering, Jiangxi Normal University, Nanchang, 330022, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 16 April 2014 Received in revised form 21 July 2014 Accepted 11 August 2014 Available online 17 August 2014
It is highly desirable to develop a simple and sensitive method for Hg2 þ detection because of the dangerous nature of Hg2 þ . In this work, we prepared a dual-emission fluorescent probe for Hg2 þ detection by combining two lanthanide chelates with different emission wavelengths. Green-emitting terbium (Tb3 þ ) chelates as reference signals were embedded into SiO2 nanoparticles and red-emitting europium (Eu3 þ ) chelates as response units were covalently linked to the surface of silica shell. Upon the addition of Hg2 þ , the fluorescence of Eu3 þ chelates can be selectively quenched, while the fluorescence of Tb3 þ chelates remained unchanged. As a kind of Hg2 þ nanosensor, the dual-emission fluorescent probe exhibited excellent selectivity to Hg2 þ and high sensitivity up to 7.07 nM detection limit. The Hg2 þ levels in drinking water and milk samples were determined by using the dual-emission fluorescent probe with satisfied recovery. Additionally, our probe has a long enough fluorescence lifetime, which can avoid the interference from autofluorescence of the biological samples. We envision that the proposed probe could find great potential applications for ultrasensitive time-resolved fluorometric assays and biomedical imaging in the future. & 2014 Elsevier B.V. All rights reserved.
Keywords: Lanthanide Dual-emission Fluorescent probe Mercury (II) Milk
1. Introduction Mercury is one of the most toxic elements in ecosystems. Contamination of the environment with mercury is an important concern throughout the world (Clarkson, 1997). It is estimated that the total global mercury emissions from all sources – both natural and human-generated – reached nearly 7500 ton per year (EPA, 2005). Water soluble divalent mercuric ion (Hg2 þ ) is one of the most usual and stable forms of mercury pollution. Because of its non-biodegradation, Hg2 þ cannot be destroyed once it was released into the environment. Hg2 þ can be absorbed by crops, then contaminate food products, such as vegetables, rice, fruit, and even dairy products (Akkemik et al., 2012). The subsequent accumulation of Hg2 þ by humans through food chain can cause the damage of brain, kidneys, nervous system, and endocrine system (Nolan and Lippard, 2008; Zalups, 2000). Therefore, monitoring of the Hg2 þ level in environmental and biological samples is an important issue to understand its distribution and potential pollution.
n
Corresponding authors. Tel./fax: þ86 791 88120861. E-mail addresses: [email protected] (H. Tan), [email protected] (L. Wang). http://dx.doi.org/10.1016/j.bios.2014.08.015 0956-5663/& 2014 Elsevier B.V. All rights reserved.
To date, many methods have been developed for the detection of Hg2 þ , such as atomic absorption spectrometry (Labatzke and Schlemmer, 2004), inductively coupled plasma mass spectrometry (Wang et al., 2007), electrochemistry (Mahajan et al., 2003) and so on. These analytical methods can measure Hg2 þ levels sensitively and accurately, but they require expensive and sophisticated equipment and/or involve tedious sample preparation procedures prior to the analysis, which limits their applications in laboratory. Fluorescent detection of Hg2 þ would be a more desirable method due to its operational simplicity, easy preparation of sample and high sensitivity. Over the past few years, some fluorescent probes based on organic molecules (Wang et al., 2006; Yoon et al., 2007; Zhu et al., 2006), conjugated polymers (Liu et al., 2007; Zhao and Zhong, 2006), quantum dots (Chen et al., 2006; Duan et al., 2009) and carbon dots (Lu et al., 2012; Zhou et al., 2012) have been applied to detect Hg2 þ . Particularly, lanthanide-based fluorescent probes for Hg2 þ detection have been paid considerable attention due to their unique optical properties including large Storks shift, sharp emission, high quantum yields and long lifetime. These properties enable the lanthanide-based fluorescent probes to eliminate efficiently the interferences from background and scattering fluorescence (Richardson, 1982). Nevertheless, few studies have centered on using lanthanide-based fluorescent probes for the detection of Hg2 þ .
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In the previous works, we reported two different methods to detect Hg2 þ using terbium (Tb3 þ ) chelate (Tan et al., 2011) and lanthanide coordination polymers (Tan et al., 2012) as fluorescent probes, respectively. The former was designed based on the fluorescence quenching of Tb3 þ chelate which resulted from the replacement of Tb3 þ by Hg2 þ , and the latter was constructed on the basis of Hg2 þ -induced fluorescence enhancement through a process of photoinduced electron transfer. Although these two lanthanide fluorescent probes exhibited high sensitivity and excellent selectivity for Hg2 þ , they were single wavelength intensity-based detection methods and tended to be affected by the concentration change of probes and/or the microenvironment of complex samples. Recently, dual-emission fluorescent probebased detection methods have received considerable attention. Compared with single wavelength intensity-based detection methods, the signal variation of dual-emission fluorescent probe is easier to be distinguished by naked eyes. Moreover, dualemission fluorescent probe may provide improved quantification accuracy because of its independence of probe concentration (Qu et al., 2013; Zhang et al., 2011). Dual-emission fluorescent probe is usually constructed by combining two fluorophores with different emission wavelengths, one fluorophore functions as reference unit and another as response moiety. Up to now, many fluorophores including organic dyes (Yilmaz et al., 2010; Zong et al., 2011), quantum dots (Zhang et al., 2011), carbon dots (Liu et al., 2014; Zhu et al., 2012), and gold cluster (Wang et al., 2014) have been employed to construct dual-emission fluorescent probes. In spite of this, the development of dual-emission fluorescent probe for Hg2 þ detection is still limited by the design of new organic fluorophores which are specific and sensitive to Hg2 þ . In this work, we attempt to employ two lanthanide chelates with different emission wavelengths to prepare a dual-emission fluorescent probe for the detection of Hg2 þ . Two lanthanide chelates were synthesized by the chemical coordination of terbium ion (Tb3 þ ) and europium ion (Eu3 þ ) with dipicolinic acid (DPA, 2.6-pyridinedicarboxylic acid), respectively (denoted as TbDPA chelate and Eu-DPA chelate). Owing to the sensitization effect of DPA, Tb-DPA chelate emits strong green fluorescence, whereas a strong red fluorescence can be observed from Eu-DPA chelate. To construct a dual-emission fluorescent probe (Tb-DPA@SiO2-EuDPA), Tb-DPA chelate was embedded into SiO2 nanoparticle as reference signal and Eu-DPA chelate was immobilized on the
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surface of silica shell as response unit. Apart from the sensitization effect, DPA is a very useful masking agent to eliminate the interference from other metal ions in various Hg2 þ sensor systems (Chen et al., 2011; Darbha et al., 2007; Huang and Chang, 2006; Liu et al., 2012). Thus, Tb-DPA@SiO2-Eu-DPA might possess the potential as a dual-emission fluorescent probe for selective detection of Hg2 þ . In addition, the surface of SiO2 nanoparticle dropped with Tb-DPA chelate was functionalized by diethylenetriaminepentaacetic acid (DTPA) to immobilize Eu-DPA chelate. DTPA was selected as the functional ligand; on the one hand, it can offer its carboxyl groups to coordinate with Eu-DPA chelate, and leading to the formation of DTPA-Eu-DPA ternary complex on surface of the SiO2 nanoparticle. On the other hand, DTPA might assist DPA to selective response to Hg2 þ due to its higher binding constant to Hg2 þ (K ¼1026.4) than Eu3 þ (K ¼1022.39) (Holloway and Reilley, 1960; Patnaik and Dean, 2004). In the presence of Hg2 þ , therefore, quenched Eu3 þ fluorescence on the surface of SiO2 nanoparticle would be observed due to the replacement of Eu3 þ by Hg2 þ (Scheme 1).
2. Material and methods 2.1. Chemicals and solutions Terbium nitrate (99.99%) and europium nitrate (99.99%) were purchased from Ruike Rare Earth Metallurgy and Functional Materials Co., Ltd. (Baotou, China); Dipicolinic acid (99%, 2, 6-pyridinedicarboxylic acid) and diethylenetriaminepentaacetic acid dianhydride (98%, DTPA dianhydride) was purchased from Sigma-Aldrich (Shanghai, China); Tetraethyl orthosilicate (99%, TEOS); 1-hexanol (99%); cyclohexane (99.5%), and metal salts (Hg(NO3)2, AgNO3, MgCl2, Pb(NO3)2, Zn(NO3)2, CdCl2, FeCl3, FeCl2, NiCl2, CoCl2, MnCl2, CrCl3 and CuCl2) were purchased from Sinopharm Chemical Reagent Company (Shanghai, China). 3-aminopropyltriethoxysilane (99%, APTES), N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) were obtained from Aladdin (Shanghai, China). Ultrapure water (18 MΩ cm; Milli-Q, Millipore) was used for the preparation of all aqueous solutions. Unless otherwise stated, all chemicals were of analytical reagent grade and were used without further purification.
Scheme 1. . Illustration of dual-emission fluorescent probe Tb-DPA@SiO2-Eu-DPA for the detection of Hg2 þ .
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2.2. Instruments The morphology of the as-prepared SiO2 nanoparticles was observed by transmission electron microscopy (TEM, JEM-2100, Japan). Fluorescence spectra and emission intensity were recorded on an LS55 luminescence spectrometer (PerkinElmer, UK). The detection solution was placed in a quartz micro-cuvette with 100 μL capacity and 2 mm lightpath. The 270-nm excitation wavelength was used for the emission spectra. A delay time of 0.05 ms and a gate time of 2 ms were used. For the emission lifetime, the fluorescence intensities at 545 nm for Tb3 þ and 615 nm for Eu3 þ were recorded under different delay times and fitted with an exponential function. UV–visible absorption spectra were recorded with a UV-3900H spectrophotometer (Hitachi, Japan). All the experiments were performed at room temperature. All error bars represent standard deviations from three repeated experiments. 2.3. Synthesis of Tb-DPA-APTES conjugate To covalently link Tb-DPA chelate to the silica matrix, Tb-DPAAPTES conjugate was synthesized. Briefly, 600 μL of 20 mM DPA aqueous solution was added to 500 μL anhydrous ethanol solution containing 9.0 mg of EDC and 2.8 mg of NHS and stirred for 30 min. Then, 100 μL of APTES was added to the mixture. After reacting for 2 h, 200 μL of 20 mM Tb(NO3)3 aqueous solutions was added under stirring. The reaction was kept for another 15 min. The obtained solution containing Tb-DPA-APTES conjugate was used for preparing SiO2 nanoparticles without further manipulation. 2.4. Preparation of amino-modified Tb-DPA doped SiO2 nanoparticles The Tb-DPA doped SiO2 nanoparticles (Tb-DPA@SiO2) were prepared in a water-in-oil (W/O) reverse microemulsion with the following procedure (Chen et al., 2007). To a microemulsion solution containing 4 mL of cyclohexane, 1 mL of n-hexanol, and 1 mL of Triton X-100 were added to 295 μL of Tb-DPA-APTES conjugate under stirring. After reacting for 30 min at room temperature, 85 μL of TEOS and 25 μL of ammonia solution (28%) were added. The reaction was continued for 24 h. The nanoparticles were isolated from the microemulsion by adding equal volume of acetone, followed by centrifuging and washing with ethanol and water several times to remove the surfactant and unreacted materials. To functionalize the as-prepared
nanoparticles with amino group, the cleaned nanoparticles were firstly dispersed in the microemulsion solution with the same components. Then, 20 μL of APTES was added to the mixture. After the reaction was carried out for 2 h, the nanoparticles were collected and washed by the same steps as above. 2.5. Modification of Tb-DPA@SiO2 with Eu-DPA chelate A quantity of 2 mg of NH2-modified Tb-DPA@SiO2 was suspended in 3 mL of PBS (phosphate buffered saline, 0.01 M, pH 7.4) and 20 mg of DTPA dianhydride was then added to this solution. After stirring for 2 h, the resulting mixture was collected and washed by centrifuging and ultrasonic dispersion. The washed nanoparticles were then dispersed in 3 mL of ultrapure water. And 300 μL of Eu-DPA chelate obtained by mixing Eu(NO3)3 and DPA aqueous solution with a molar ratio of 1:3 was added. The reaction was incubated for 30 min at room temperature. Those unbonded Eu-DPA chelates were removed by centrifuging. Finally, the precipitate was dispersed in 3 mL of ultrapure water to form Tb-DPA@SiO2-Eu-DPA suspension and stored 4 °C for further use. 2.6. Detection of Hg2 þ in aqueous solution For Hg2 þ detection, solutions with Hg2 þ concentrations from 0 to 10 μM were added into 10 μL of Tb-DPA@SiO2-Eu-DPA suspension, and H2O was added till the total volume reached to 100 μL. After reacting for 10 min, the fluorescence spectra were recorded using an excitation wavelength of 270 nm. To test the selectivity of Tb-DPA@SiO2-Eu-DPA to Hg2 þ , 6 μL of 10 mM stock solutions of interference metal ions was added into 10 μL of Tb-DPA@SiO2-Eu-DPA suspension, respectively. The total volume was reached to 100 μL by adding ultrapure water. The reaction was lasted 10 min before measuring the fluorescence intensities of these mixtures at 615 nm. 2.7. Detection of Hg2 þ in drinking water and milk The drinking water and milk samples were obtained from a local supermarket. To detect Hg2 þ in drinking water, different volumes of standard solutions of Hg2 þ were spiked into drinking water to prepare water samples containing different concentrations of Hg2 þ . Then, 10 μL of Tb-DPA@SiO2-Eu-DPA suspension was added to these water samples, respectively. After reacting for 10 min at room temperature, the fluorescence intensities at 615 nm of these mixtures were recorded under a 270-nm excitation wavelength. For the detection of Hg2 þ in milk, milk powders
Fig. 1. TEM images of Tb-DPA@SiO2-Eu-DPA SiO2 nanoparticles.
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were treated in following steps. Firstly, the proteins in milk powders were removed by adding 1% (v/v) trichloroacetic acid and sonicating for 20 min. Then the lipids were removed by collecting the supernatant filtered through a 0.22 mm membrane (Whatman) after centrifuging the sample at 12,000 rpm (14,463g) for 10 min. A series of milk samples containing different concentrations of Hg2 þ in the range of 0–1 μM was prepared by “spiking” them with different volumes of a stock solution of Hg2 þ (100 μM). Tb-DPA@SiO2-Eu-DPA suspension was then added to these milk samples, respectively; the total volume is 100 μL as mentioned above. The resulting solutions were mixed well and stood for 10 min before recording their emission spectra.
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between Tb-DPA chelate and Eu-DPA chelate was not occurred. Furthermore, it can be seen from Fig. 2 that the fluorescence of EuDPA chelate of the dual-emission probe showed a significant decrease upon the addition of Hg2 þ . By contrast, no marked changes (o5%) were found in the fluorescence of Tb-DPA chelate of the probe in the presence of Hg2 þ . The results suggest that the added Hg2 þ only quench the fluorescence of Eu-DPA chelates on the surface of silica shell and has no influences on the fluorescence of Tb-DPA chelate embedded into the silica core. Therefore, Tb-DPA@SiO2-Eu-DPA appears to be a useful dual-emission fluorescent probe for Hg2 þ detection. 3.2. Lifetime and absorption spectra
3. Results and discussion 3.1. Characterizations As shown in Fig. 1, the resultant SiO2 nanoparticles are uniform and monodispersed and have average sizes of 60 75 nm. The surface of the nanoparticle is rough, which may result from the conjugation of Eu-DPA chelates to the DTPA on the surface of silica shell. Moreover, a silica shell with thickness of approximately 10 nm can be observed. The existence of the silica shell is favorable to avoid the occurrence of fluorescence resonance energy transfer (FRET) process from Tb-DPA chelate to Eu-DPA chelate and to prevent the direct contact of the Tb-DPA chelate in the interior of SiO2 nanoparticles with the external solvents. Fig. 2 showed the fluorescent emission spectra of Eu3 þ functionalized Tb-DPA@SiO2 nanoparticles (Tb-DPA@SiO2-Eu3 þ ) under different conditions. In the absence of DPA, Tb-DPA@SiO2-Eu3 þ exhibited a typical emission of Tb3 þ with peaks at 490, 545, 584 and 620 nm. In the presence of DPA, however, two new emission peaks at 590 and 615 nm were observed except the existed emission peaks of Tb3 þ at 490 and 545 nm. The two new peaks can be assigned to 5D0 to 7 F1 and 5D0 to 7F2 electronic transitions of Eu3 þ , respectively (Richardson, 1982). The appearance of the emission peaks of Eu3 þ implies the occurrence of the chemical coordination of the added DPA with Eu3 þ on the surface of Tb-DPA chelates-doped SiO2 nanoparticles, which results in the formation of the dual-emission fluorescent probe Tb-DPA@SiO2-Eu-DPA. It was also noteworthy that no obvious changes in the fluorescence of Tb-DPA chelate as reference signals were observed after the formation of Eu-DPA on the surface of silica shell. This indicates that the FRET process
To understand the interaction of Hg2 þ with the dual-emission fluorescent probe, we investigated the changes of the emission lifetime of Tb-DPA@SiO2-Eu-DPA in the absence and presence of Hg2 þ . After the addition of Hg2 þ , the emission lifetime of Eu-DPA chelate on the surface of silica shell was decreased from its original 1.169 to 0.788 ms (Fig. S1). However, the emission lifetime of Tb-DPA chelate remained unchanged (data not shown). The results confirm that Hg2 þ only reacts with Eu-DPA chelate and displays no effects on the inherent transition processes of Tb3 þ induced by the ligand-field. In addition, the effects of Hg2 þ on the absorption spectra of the Tb-DPA@SiO2-Eu-DPA were examined. As shown in Fig. S2, Tb-DPA@SiO2-Eu-DPA alone exhibited an obvious absorption peak at 272 nm, which could be assigned to the maximum absorption of DPA molecules. Upon the addition of Hg2 þ , no marked changes in the absorption spectra of Tb-DPA@SiO2-Eu-DPA were observed except a slight change in the absorption intensity. Even when Hg2 þ concentration was increased to 1 mM, the absorption spectrum of Tb-DPA@SiO2-EuDPA was still not changed. The unchanged absorption spectra reflect that Eu3 þ co-coordinated to DTPA and DPA was replaced by Hg2 þ , which led to the formation of DTPA-Hg-DPA ternary complex on the surface of the silica shell. 3.3. Reaction time of Hg2 þ In order to determine the response rate of fluorescent signal upon the addition of Hg2 þ , time-dependent measurements on the fluorescence of Tb-DPA@SiO2-Eu-DPA to Hg2 þ with different concentrations were conducted. As shown in Fig. S3, with the addition of Hg2 þ , the fluorescent intensity of Tb-DPA@SiO2-EuDPA at 615 nm was rapidly decreased, and reached a steady value within 5 min. The decrease of the fluorescence intensity of Tb-DPA@SiO2-Eu-DPA at 615 nm is proportional to the concentration of Hg2 þ . This indicates that the quenching reaction of Hg2 þ to Tb-DPA@SiO2-Eu-DPA needs 5 min to complete and the decrease in the fluorescent intensity of Tb-DPA@SiO2-Eu-DPA at 615 nm is time-dependent. Thus, 5 min was chosen as the reaction time in the subsequent experiments to obtain the best sensitivity. 3.4. Detection sensitivity for Hg2 þ
Fig. 2. Emission spectra of Tb-DPA@SiO2-Eu3 þ (a), Tb-DPA@SiO2-Eu3 þ in the presence of DPA (b) and Tb-DPA@SiO2-Eu3 þ with the addition of DPA and 0.5 μM of Hg2 þ (c). The emission spectra were recorded at 270 nm excitation wavelength.
To evaluate the detection sensitivity of Tb-DPA@SiO2-Eu-DPA as a dual-emission fluorescent probe for Hg2 þ , the fluorescent responses of the probe to Hg2 þ with different concentrations were investigated. As shown in Fig. 3a, the fluorescence intensity of the probe at 615 nm was highly sensitive to Hg2 þ and decreased with the increase of Hg2 þ concentration. Nevertheless, the fluorescent intensity of the probe at 545 nm showed almost no changes. There is a good linear correlation between the intensity ratio of F545/F615 and the concentration of Hg2 þ in the range of 10 nM–2 μM (Fig. 3b). The detection limit is calculated to be 7.07 nM, which is lower than the maximum level (10 nM) of
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a
b
Fig. 4. The detection selectivity of Tb-DPA@SiO2-Eu-DPA in the presence of 6 μM various metal ions. Black bars represent the addition of single metal ion (6 μM); Red bars represent the addition of single metal ion (6 μM) and Hg2 þ (6 μM) together except Fe3 þ . The gray and red bars of Fe3 þ represent the addition of mixture of Fe3 þ and H2O2 in the absence and presence of Hg2 þ , respective. Relative standard deviations (RSD) are all less than 7.41%. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
displays excellent fluorescence stability at room temperature. No obvious changes in the fluorescent response of the dual-emission probe to Hg2 þ were observed after the coordination polymers were stored for 30 days (Fig. S4). This suggests that the dualemission probe possesses great potential as a fluorescence material to detect Hg2 þ . 3.5. Detection selectivity for Hg2 þ
c
Fig. 3. (a) Fluorescence emission spectra of Tb-DPA@SiO2-Eu-DPA in the presence of different concentrations of Hg2 þ solution. (b) The plot of F545/F615 as a function of the Hg2 þ concentration. Relative standard deviations (RSD) are all less than 4.39%. (c) Visual fluorescence color changes of the dual-emission fluorescent probe upon the addition of different concentrations of Hg2 þ (from left to right: 0, 0.04, 0.2, 1, 6 μM). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Hg2 þ in drinking water permitted by the U.S. Environmental Protection Agency (EPA) (EPA, 1988). Compared with other fluorescent methods for Hg2 þ detection (Chen et al., 2011; Duan et al., 2009, 2014; Mandal et al., 2013; Yan et al., 2013; Zhou et al., 2012), the presented dual-emission probe displayed a comparable detection limit. Additionally, Hg2 þ -induced fluorescence changes can be observed under a UV lamp. From Fig. 3c, it can be seen that the color of the dual-emission probe solution gradually changed from orange to green upon the titration with Hg2 þ , which enabled the naked-eye to detect Hg2 þ . Besides, the dual-emission probe
To evaluate the selectivity of the dual-emission probe for Hg2 þ , we investigated the response of the probe to other metal ions that often coexisted with Hg2 þ in the environment. These metal ions employed in our selective experiments included Ca2 þ , Cd2 þ , Co2 þ , Cr3 þ , Cu2 þ , Fe2 þ , Fe3 þ , K þ , Na þ , Mg2 þ , Mn2 þ , Ni2 þ and Pb2 þ . From Fig. 4, it can be found that Hg2 þ was the only metal ion which caused a significant change in F545/F615 of Tb-DPA@SiO2-EuDPA except Fe3 þ , and no obvious fluorescent responses can be found in the presence of the interfering metal ions. The interference of Fe3 þ can be eliminated by the addition of H2O2 because H2O2 can reduce Fe3 þ to Fe2 þ . Since the fluorescence of Tb-DPA@SiO2-Eu-DPA stays constant in the presence of H2O2 with different concentrations (data not shown), H2O2 has no effects on the detection of Hg2 þ . The selective response of Tb-DPA@SiO2-EuDPA to Hg2 þ can be attributed to the higher binding ability of DTPA to Hg2 þ than other interfering metal ions (Holloway and Reilley, 1960; Patnaik and Dean, 2004) and the selective recognition ability of DPA to Hg2 þ (Darbha et al., 2007; Huang and Chang, 2006). In addition, the potential effects of the interfering metal ions on the detection of Hg2 þ were assessed by examining the fluorescence changes of Tb-DPA@SiO2-Eu-DPA in the presence of 6 μM Hg2 þ and interferential metal ions (each 6 μM), respectively. The results were displayed in Fig. 4 (red bars), which indicates that the coexistence of these interfering metal ions has virtually no influence on the detection of Hg2 þ and further demonstrated the high specificity of the dual-emission probe for Hg2 þ . Due to the fact that milk might contain some substances that would affect the fluorescence of Eu-DPA chelate on the surface of silica shell by coordinating with Eu3 þ , such as amino acids, glucose and vitamins, the fluorescent responses of the probe to these substances were then studied. As shown in Fig. S5, no remarkable fluorescence responses were observed upon the
H. Tan et al. / Biosensors and Bioelectronics 63 (2015) 566–571
addition of these amino acids (Arg, Tyr, Asp, Glu, His, Lys, Cys), ascorbic acid, fructose, lactose, glucose, Vit B1 and Vit B12; only Hg2 þ can cause the decrease of the fluorescence of Tb-DPA@SiO2 -Eu-DPA at 615 nm. The results suggest that the influences from these foreign substances are negligible, and the presented dualemission probe can be used for the detection of Hg2 þ in milk sample with high selectivity.
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Scientist Foundation of Jiangxi Province (20112BCB23006 and 20122BCB23011).
Appendix A. Supplementary information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.08.015.
3.6. Detection of Hg2 þ in drinking water and milk samples The presented dual-emission probe was further employed to determinate the levels of Hg2 þ in drinking water and milk samples. To detect Hg2 þ in drinking water and milk samples, standard addition method was used. As shown in Table S1, the recoveries of Hg2 þ in drinking water were between 97.2% and 105.4%, while for milk samples they were between 97.05% and 114.82%. The relative standard deviations (RSD, n ¼3) are all less than 2.35%. The results indicate that the dual-emission probe has good precision and satisfactory reproducibility. It is noteworthy that our analysis works admirably in highly low matrix samples that are not representative of real contaminated food.
4. Conclusion In summary, we have prepared a lanthanide-based dual-emission fluorescent probe for Hg2 þ detection by employing Tb-DPA and Eu-DPA chelates as reference signal and response moiety, respectively. The dual-emission fluorescent probe exhibited excellent selectivity to Hg2 þ and high sensitivity up to 7.07 nM detection limit. Compared with other dual-emission fluorescent methods, our fluorescent probe displayed a comparable detection limit and possessed a long enough fluorescence lifetime for the time-resolved fluorescence assays, which was especially advantageous for the detection of biosamples with autofluorescence. To the best of our knowledge, this is the first report on using two lanthanide chelates with different emission wavelengths to construct a dual-emission fluorescent probe. Furthermore, the resulted fluorescent probe was employed to detect the level of Hg2 þ in drinking water and milk samples and satisfactory results were obtained. We believe that the proposed strategy can be extended to the preparation of other lanthanide-based dual-emission fluorescent probes for the wide applications in biosensing, imaging, environmental analysis and so on.
Acknowledgment This work was supported by National Natural Science Foundation of China (Nos. 21305054 and 21165010), Scientific Research Foundation of Jiangxi Normal University, Specialized Research Fund for the Doctoral Program of Higher Education of China (No. 20133604120002), Scientific Research Foundation of Education Commission of Jiangxi Province (No. GJJ14258), and Young
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