Talanta 132 (2015) 864–870
Contents lists available at ScienceDirect
Talanta journal homepage: www.elsevier.com/locate/talanta
A highly selective and sensitive fluorescent probe for quantitative detection of Hg2 þ based on aggregation-induced emission features Aizhi Wang, Yunxu Yang n, Feifei Yu, Lingwei Xue, Biwei Hu, Weiping Fan, Yajun Dong Department of Chemistry and Chemical Engineering, School of Chemistry and Biological Engineering, University of Science and Technology Beijing, Beijing 100083, China
art ic l e i nf o
a b s t r a c t
Article history: Received 31 July 2014 Received in revised form 19 October 2014 Accepted 22 October 2014 Available online 31 October 2014
A π-conjugated cyanostilbene derivative of (Z)-2-(4-nitrophenyl)-3-(4-(vinyloxy)phenyl)acrylonitrile (CN-vinyl) had been designed, synthesized and confirmed by the standard spectroscopic analyses. CN-vinyl possesses an unusual high emissive aggregation-induced emission (AIE) feature in a tetrahydrofuran/water mixture (2:8, v/v). The fluorescence intensity of CN-vinyl can be quenched linearly with the addition of Hg2 þ in a range of 0–50 μM with a correlation coefficient of R2 ¼0.9957. The detection limit of Hg2 þ is 37 nM. The mechanism for Hg2 þ -mediated optical properties of CN-vinyl is due to the selective cleavage of vinyl group by Hg2 þ . Accuracy of the proposed methodology was evaluated by means of the recovery study in real samples and the analyzing certified reference material of the standard solution of Hg2 þ . By this novel strategy, CN-vinyl can be used for quantitative detection of Hg2 þ as well as the presence of other physiological relevant metal ions. & 2014 Elsevier B.V. All rights reserved.
Keywords: Fluorescent probe Quantitative detection Mercury ion Aggregation-induced emission
1. Introduction Mercury, a class of heavy metal distributed throughout the environment by both natural processes and human activities, has long been identified as one of the most toxic heavy metals and a kind of persistent contaminant [1,2]. It can first be transformed into organic mercury after being absorbed from the environment by organisms, and then accumulated highly in trophic animals through the food chain [3,4]. According to recent reports, the toxicity of Hg2 þ can induce a variety of syndromes and harmful effects [5], such as damage DNA, disable normal functions of liver and kidney, disrupt the immune system homeostasis [6–9] etc. Therefore, efficient monitoring the trace amount of Hg2 þ with highly selective and sensitive is remarkably important for human health. Conventional analytical techniques include spectrophotometry, atomic absorption spectrometry, inductively coupled plasma–mass spectroscopy (ICP–MS), cold-vapor atomic fluorescence spectrometry (CV-AFS), ultraviolet–visible spectrometry, and X-ray absorption spectroscopy [10–15]. Whereas, these methods require expensive intricate instrumentation and complicated sample preparation processes. Moreover, the complexity makes them unsuitable for using in on-site Hg2 þ analyses. Different from the traditional Hg2 þ analyses, fluorescence probe offers several advantages for Hg2 þ measurements because of its high sensitivity, lower detection limit,
n
Corresponding author. Tel.: þ 86-010-62333871 E-mail address: [email protected] (Y. Yang).
http://dx.doi.org/10.1016/j.talanta.2014.10.048 0039-9140/& 2014 Elsevier B.V. All rights reserved.
non-sample destructing and simple operation. In consequence, these unique characteristics make it favorable for both detection and imaging of Hg2 þ in biological samples [16]. Many Hg2 þ chemosensors with fluorescence response have been reported, such as, proteins conjugated polymer and diblock copolymer fluorescence sensors, biomolecules fluorescence sensors, foldamer-based fluorescence sensors, inorganic gold nanoparticles and CdS nanoparticles, and small organic molecule fluorescence probes [9,17–20]. However, luminescence of these conventional fluorophores often becomes weakened or quenched at high concentration or solid state due to the thermodynamically favorable process under the driving forces of intermolecular π–π stacking interaction of aggregation. The outcome of this effect is the sacrifice of the fluorescence emission efficiency and reduction of sensing performance. Fortunately, in 2001, Tang and co-workers first observed an intriguing phenomenon [21] that is the exact opposite of the aggregation-caused quenching (ACQ). Some practically nonluminescent in the solution state but become strongly emissive when aggregated or in the solid state due to suppression of nonradiative deactivation associated with restriction of intramolecular rotations (RIR). Then a large number of so called “aggregation-induced emission (AIE)” active molecules have since been developed, example of which including siloles, 1-cyano-trans-1, 2-bis-(40 -methylbiphenyl) ethylene (CN-MBE), 2, 5-diphenyl-1, 4-distyrylbenzene (DPDSB) derivatives, fluorenonearylamine derivatives, diphenyldibenzofulevne (DPDBF) derivatives, conjugated polymers, and so on [22–29]. Most of them can be employed as optical devices or bioprobes.
A. Wang et al. / Talanta 132 (2015) 864–870
865
Infrared spectra were recorded on a Shimadzu IR-8400S spectrophotometer. 1H NMR and 13C NMR spectra were recorded at 400 MHz on a Varian Gemin-400 respectively. ESI-MS spectra were obtained on Bruker spectrometers. All absorption and fluorescence spectra in this work were recorded in Pgeneral TU-1901 and Hitachi F-4500 fluorescence spectrometers. All pH values were measured with a Model pHS-3C pH meter (Shanghai, China). Analytical TLC was carried out on silica gel plates (HSGF254, 0.2 mm, Yantai Chemical Industry Research Institute). Melting points were measured with RD-II melting point apparatus (uncorrected, Tainjin Xintianguang Instrument Company).
Scheme 1. Synthesis of CN-vinyl.
Scheme 2. The reaction mechanism of CN-vinyl with Hg2 þ .
Zhu et al. have demonstrated that the vinyl group is useful to design Hg2 þ probe [30]. Inspired by their works, a novel fluorescence turn-off probe CN-vinyl ((Z)-2-(4-nitrophenyl)-3-(4-(vinyloxy)phenyl) acrylonitrile) toward Hg2 þ was designed and synthesized (as shown in Scheme 1). CN-vinyl is a potential AIE cyanostilbene derivatives based on π-conjugated fluorophore, and the vinyl group as a recognition site with unique and high reactivity toward Hg2 þ . We hypothesized that the reaction of CN-vinyl with Hg2 þ would result in the cleavage of the vinyl group and the recognition mechanism is shown in Scheme 2. In addition, compound-2 (Z)-3-(4-hydroxyphenyl)-2-(4-nitr-ophenyl)acrylonitrile was also synthesized as a contrast of CN-vinyl and the synthetic route is shown in Fig. S1.
2. Experimental section
2.2. Synthesis 2.2.1. Synthesis of 4-(2-bromoethoxy)benzaldehyde (compound-3) A similar method was adopted to synthesize the titled compound according to literature [31]. To a solution of p-hydroxybenzaldehyde (1.22 g, 0.01 mol) and anhydrous K2CO3 (4.14 g, 0.03 mol) in 20 ml DMF, requisite linear chain aliphatic dibromoethane (9.35 g, 0.05 mol) was added and then stirred at 80 1C for 1 h. After the reaction was finished (controlled by TLC), the reaction mixture was cooled to 0 1C and quenched with 1 M HCl. The mixture was then extracted with CHCl3 and the organic layer was washed with water, dried over anhydrous Na2SO4, and then filtered out. Excess solvent was evaporated under reduced pressure and the resultant solid left was purified by column chromatography (EtOAc/petroleum ether¼1:5 as eluent) to afford compound-3 (1.63 g, 72% yield). mp 52–54 1C. IR (KBr) v: 1685, 1602, 1577, 1506, 1253, 1163, 831 cm 1. 1 H NMR (400 MHz, CDCl3) δ: 9.88 (s, 1H), 7.83 (d, J¼ 8.8 Hz, 2H), 7.00 (d, J¼ 8.8 Hz, 2H), 4.36 (t, J¼ 6.4 Hz, 2H), 3.65 (t, J¼6.4 Hz, 2H).
2.2.2. Synthesis of 4-(vinyloxy)benzaldehyde (compound-4) A similar method was adopted to synthesize the titled compound according to the literature [32]. To a solution of compound-3 (0.227 g, 0.001 mol) in DMSO (10 mL), t-BuOK (0.224 g, 0.002 mol) was added. The reaction mixture was stirred at 25 1C for 4 h, after which it was diluted with EtOAc (20 mL), washed with H2O (3 10 mL) and then brine (20 mL). The organic layer was dried, filtered, and concentrated. The crude product was purified by column chromatography (EtOAc/petroleum ether¼1:5 as eluent) to afford compound-4 as a colorless oil (0.082 g, 56% yield). IR (KBr) v: 1697, 1645, 1598, 1504, 1299, 1249, 1161, 958, 833 cm 1. 1H NMR (400 MHz, CDCl3) δ (ppm): 9.90 (s, 1H), 7.85 (d, J¼8.8 Hz, 2H), 7.09 (d, J¼ 8.8 Hz, 2H), 6.67 (dd, J¼ 6.0 Hz, 13.6 Hz, 1H), 4.92 (dd, J¼2.0 Hz, 13.6 Hz, 1H), 4.61 (dd, J¼ 1.6 Hz, 6.0 Hz, 1H).
2.1. Reagents and apparatus All reagents and solvents were commercially available and were used without further purification unless otherwise stated. The water used for investigating was distilled prior to use. 4-nitrobenzylcyanide and p-hydroxybenzaldehyde were purchased from Alfa Aesar. MeHgCl was purchased from Dr. Ehrenstorfer Germany. The certified reference material of the standard solution of Hg2 þ was purchased from General Research Institute for Nonferrous Metals, China and the certificate information is shown in Table S1. Stock solutions of metal ions (2.5 mM, Li þ , Na þ , K þ , Mg2 þ , Ca2 þ , Al3 þ , Fe2 þ , Fe3 þ , Mn2 þ , Cu2 þ , Co2 þ , Ni2 þ , Pb2 þ , Zn2 þ , Sn2 þ , Ag þ , Cd2 þ , Ba2 þ , Cr3 þ , MeHg þ , Hg2 þ ) were prepared in deionized water. Stock solution of CN-vinyl (2.5 mM) was prepared by dissolving 18.2 mg CN-vinyl in 25 ml THF. Phosphate buffer solutions (PBS) were prepared by combining proper amounts of NaH2PO4 and NaOH and adjusted by a pH meter. Purification of reaction products was carried out by flash chromatography using silica gel (300–400 mesh, Branch of Qingdao Haiyang Chemical Co., Ltd.).
2.2.3. Synthesis of (Z)-2-(4-nitrophenyl)-3-(4-(vinyloxy)phenyl) acrylonitrile (CN-vinyl) To a mixture of compound-4 (0.148 g, 0.001 mol) and 4-nitrobenzylcyanide (0.162 g, 0.001 mol) in absolute EtOH (5 mL), piperidine (0.255 g, 0.003 mol) was added with dropwise. After the reaction mixture was stirred at room temperature for 3 h, the obtained precipitated was filtered, washed with EtOH, dried and purified by column chromatography (CH2Cl2/petroleum¼1:2 as eluent), a yellow solid was obtained. (0.233 g, 79% yield). mp 175–177 1C. IR (KBr) v: 2212, 1643, 1596, 1579, 1508, 1336, 1257, 1180, 848 cm 1. 1H NMR (400 MHz, DMSO-d) δ (ppm): 8.34 (d, J¼8.8 Hz, 2H), 8.28 (s, 1H), 8.06–8.00 (m, 4H), 7.27 (d, J¼8.8 Hz, 2H), 7.03 (dd, J¼6.0 Hz, 13.6 Hz, 1H), 4.89 (dd, J¼1.6 Hz, 13.6 Hz, 1H), 4.62 (dd, J¼ 1.6 Hz, 6.0 Hz, 1H). 13 C NMR (DMSO) δ (ppm): 158.9, 147.6, 147.3, 146.06, 140.7, 132.5, 132.3, 130.6, 128.3, 127.2, 124.8, 118.0, 117.1, 116.7, 106.7, 97.4, 97.3. ESIMS (m/z): calcd for: C17H12N203 [MþCl] 327.05, found 327.1. Anal. calcd for: C17H12N2O3, C, 69.86; H, 4.14; N, 9.58; found: C, 70.55; H, 4.07; N, 9.56.
A. Wang et al. / Talanta 132 (2015) 864–870
To characterize the AIE property of CN-vinyl, 100 μL stock solution of CN-vinyl (25 mM) was added to 10 mL THF/water (10:0, 4:6, 3:7, 2:8, 1:9, v/v, containing 50 mM NaH2PO4–NaOH). As shown in Fig. S10, the effect of water volume fraction on the pH was measured in THF/water containing 50 mM PBS. When THF and PBS was mixed well, the acidity of the solution decreased slightly (pH¼7.21–7.94) compared with PBS (pH ¼7.0). It is probably due to the restricting ionization of acid induced by THF. Then, the mixed solution was incubated at room temperature for 15 min and was transferred to a 1 cm quartz cell for absorption spectra and fluorescence spectra. In a standard detection method of Hg2 þ , the test solution was prepared by placing 100 μL stock solution of CN-vinyl and 1.9 ml THF into a test tube, diluted to 10 mL with 50 mM PBS at pH ¼7.0, added appropriate amount of Hg2 þ ions, incubate at room temperature for 15 min, and then record the fluorescence spectra. To evaluate any possible facts which may affect the response of the fluorescence intensity caused by interfering ions, three sequences of addition were attempted. (1) The interfering metal ions were added to the test solution, and then Hg2 þ was added to explore whether the reaction of CN-vinyl with Hg2 þ ions was affected by the interfering metal ions; (2) Hg2 þ ions were added to the test tube ahead, and then other interfering metal ions were added to explore whether the fluorescence intensity of reaction product was effected by interfering metal ions; (3) Hg2 þ ions and interfering metal ions were added to the test tubes at the same time to simulate the application in real sample analysis. All of them were allowed to incubate at room temperature for 15 min following to record the fluorescence spectra. For application in real sample analysis, the concentration of Hg2 þ in real samples was detected by the standard addition method. HgCl2 (4.1 mg) was added to 500 ml volumetric flask and diluted to metered volume with three real water samples which contain 50 mM PBS at pH ¼ 7.0 respectively, and gave 30 μM real sample solutions of Hg2 þ . Moreover, the certified standard solution of Hg2 þ (2.5 ml) was transferred to a 500 ml volumetric flask and diluted with PBS (50 mM, pH ¼ 7.0) to the metered volume, and give a 25 μM standard solution of Hg2 þ . Subsequently, 100 μL stock solutions of CN-vinyl and 1.9 ml THF were placed into test tubes and diluted to 10 mL with the three real samples and the standard solution of Hg2 þ respectively, and gave the test solutions. Finally, the fluorescence emission spectra were recorded. All fluorescence spectra were recorded in the range from 480 nm to 650 nm using a 456 nm excitation wavelength. The excitation and emission bandwidths were set to 5 nm and 10 nm respectively.
3. Results and discussion 3.1. Aggregation-induced emission characteristics of CN-vinyl We first studied the optical properties of CN-vinyl and compound-2. As shown in Fig. S11, both the absorption spectra of CNvinyl and compound-2 in THF solution exhibited three absorption bands at 245.0, 291.5, 365.5 nm and 249, 293.5, 368 nm respectively. Although CN-vinyl has a longer π-conjugated unit, it showed a blueshift spectrum due to a more twisted conformation caused by the cyano and vinyl group. This process had been supported by the theoretical calculations which was performed at B3LYP/6–31G (d) basis in the Gaussian 09 package. The optimized molecular structures and HOMO and LUMO energy levels of CN-vinyl and compound-2 are shown in Fig. S12. Obviously, both of them have
strong intramolecular charge transfer (ICT) effect from the electron cloud distribution. The calculated energy band gap for CN-vinyl is 3.456 eV, which is wider than that of compound-2 (3.392 eV). Hence, the results of theoretical studies are consistent with the absorption spectra. The characteristics of CN-vinyl were investigated in THF/water (from 10:0 to 1:9, v/v) phosphate buffer solution (PBS, 50 mM, pH ¼7.0) and the results are shown in Figs. 1 and 2. As expected, there was a structured absorption spectra and almost no fluorescence emission could be detected in dilute THF solution, suggesting that CN-vinyl is non-fluorescence when dispersed in its “solution” state. However, its fluorescence spectra in a THF/water mixture with a high water fraction (80%) had an intensive band at 537 nm, and showed a leveling-off in the ultraviolet region of the absorption spectra (commonly observed in nanoaggregates suspension). The fluorescence increased by 79 times compared with that in pure THF and a lower THF volume fraction was found to enhance the AIE fluorescence intensity. These results correlated well with the fact that more aggregates were formed in poorer solvents. On the other hand, the maximum peak in the absorption spectra of the nanoparticle suspension of CN-vinyl had a clearly red-shift (12 nm) compared with that of dilute solution (as shown in Fig. 2). This result implied that there is an extension of the effective conjugation length which was caused by the planarization of twist molecular in nanoparticles. The new shoulder band (the arrow) observed around 450 nm should be resulted from the
Fluorescence intensity (a.u.)
2.3. Analytical procedure
600
THF
500
60% Water 70% Water
400
80% Water
300
90% Water 200
100
0 480
530
580
630
Wavelength (nm)
600 500 Fluorescence intensity (a.u.)
866
400 300 200 100 0 0
20
40
60
80
100
Water fraction (%)
Fig. 1. (a) Fluorescent spectra of CN-vinyl (25 μM) in a THF/water mixture (excitation wavelength was 456 nm) (b) Effect of water volume fraction on the emission intensity of CN-vinyl (25 μM) in THF/water containing 50 mM PBS at pH 7.0. Inset show photographs of CN-vinyl (25 μM) in dilute THF solution and a THF/ water mixture with a high water fraction under a UV lamp (365 nm). Excitation and emission was at 456 nm/537 nm respectively.
A. Wang et al. / Talanta 132 (2015) 864–870
867
3.3. Fluorescence detection of Hg2 þ based on CN-vinyl
Fig. 2. Absorption spectra of CN-vinyl (25 μM) in a THF/water mixture.
450
CN-vinly
Fluorescence intensity (a.u.)
400 350
2+
CN-vinly + Hg
300 250
The fluorescence spectra of CN-vinyl upon titration with Hg2 þ was recorded in THF/water (2:8, v/v) buffered by 50 mM PBS at pH¼7.0 to investigated the quenching behavior of Hg2 þ ion. As shown in Fig. 4, CN-vinyl itself showed a very strong emission. However, the fluorescence of CN-vinyl (25 μM) at 537 nm was dramatically decreased upon the addition of Hg2 þ and followed a slight red-shift. Simultaneously, a perceived fluorescence color change of CN-vinyl was observed from yellow to colorless (Fig. 4, insert). It was plausible to suppose that a water-soluble species (should be compound-2) increased and the formation of aggregation of CN-vinyl decreased with the cleavage of the vinyl group upon the addition of Hg2 þ to CN-vinyl. The quenching of photoluminescence is observed at a lower concentration of Hg2 þ . The change of emission intensity became constant and caused 7-fold decrease when the addition of Hg2 þ added to 2.0 equiv. (50 μM). There was an approximately linear relationship (R2 ¼ 0.9957) between fluorescence intensity at 537 nm and Hg2 þ concentration in the range of 0 to 50 μM (Fig. 5). The detection limit was found to be 37 nM on the basis of the equation LOD ¼ 3δ/m (δ was the standard deviation of the blank solution and m is the absolute value of the slope between fluorescence intensity and Hg2 þ concentration) (Fig. S16). Above results demonstrated that our
200 150 100 50 0 1
2
3
4
5
6
7
8
9
10
11
12
13
pH
Fig. 3. Effect of pH on the emission intensity of probe CN-vinly (25 μM) at 537 nm in the absence and presence of 50 μM of Hg2 þ . All measurements were taken in PBS (50 mM, pH¼ 7.0) at25 1C. Excitation and emission were at 456 nm/537 nm respectively.
J-type aggregation. These date clearly indicated that CN-vinyl exhibits a significant feature of AIE.
The reaction conditions, such as pH, incubation time and buffer capacity of PBS, play an important role in the sensing process. The effects of pH value on the fluorescence intensity of CN-vinyl (25 μM), and the mixture of CN-vinyl (25 μM) with Hg2 þ (50 μM) in THF/ water (2:8, v/v) were investigated at first. As shown in Fig. 3, the fluorescence intensity of CN-vinyl decreased gradually with the rising of pH from 7.0 to 1.0 due to the formation of oxonium ion. However, when pH increased up to 10, there was almost no fluorescence because of the 1, 4-addition reaction of OH- to α, β-unsaturated cyano compound, which lead to destruction of the conjugate structure. As CN-vinyl displayed a strong fluorescence changes in the pH range of 6–9 upon the addition of Hg2 þ , and considering the application of samples in physiological or environmental, pH¼7.0 was thus chosen as the experiment condition. In addition, the results of timedependent fluorescence measurement of CN-vinyl (25 μM) and Hg2 þ (50 μM) in THF/water (2:8, v/v, PBS, pH¼7.0) is displayed in Fig. S13, of which would be quenched completely after 15 min. So, all the results of the assay were tested at the time of 15 min after the CN-vinyl was mixed with the Hg2 þ . Finally, the buffer capacity of PBS (pH¼7.0) was explored. As shown in Figs. S14 and 15, we could see that pH present a relatively stable value even if the concentration of Hg2 þ ions or other interfering metal ions reach its maximum (50 μM) in 50 mM PBS.
Fig. 4. Fluorescence spectra of CN-vinyl (25 μM) in the presence of different concentrations of Hg2 þ ion at a THF/water mixture (2:8, v/v); Inset: fluorescence color change of CN-vinyl (25 μM) in the absence and presence of Hg2 þ (2.0 equiv.). All measurements were taken in PBS (50 mM, pH 7.0) at 25 1C. Excitation wavelength was 456 nm.
450 400
Fluorescence intensity (a.u.)
3.2. Optimization of the analytical condition
350 300 y = -159.48x + 361.85 R² = 0.9957
250 200 150 100 50 0 0
0.5
1
1.5
2
2.5
3
3.5
equiv. of Hg2+
Fig. 5. The relationship between fluorescence intensity and Hg2 þ concentration. Plots of the fluorescence intensity obtained from the reaction of CN-vinyl (25 μM) with Hg2 þ (0–2.0 equiv.).All measurements were taken in PBS (50 mM, pH 7.0) at 25 1C. Excitation and emission were at 456 nm/537 nm respectively.
A. Wang et al. / Talanta 132 (2015) 864–870
proposed CN-vinyl could detect Hg2 þ in quantitative and with excellent sensitivity by the fluorescence spectrometry method. 3.4. Reaction mechanism It has been reported that Hg2 þ could selectively cleave the vinyl group by the oxymercuration reaction [33]. To verify our design principle of CN-vinyl for Hg2 þ , the reaction mechanism was studied by 1H NMR and mass spectra. Compound-2 was isolated from the reacting system to confirm that the breakage action of CN-vinyl with Hg2 þ had indeed produced after Hg2 þ was added to CN-vinyl. 1H NMR spectra of CN-vinyl, the isolated reaction product of compound-2 and the synthesized compound-2 were shown in Fig. 6A–C. Upon the addition of Hg2 þ , three protons of CN-vinyl corresponding to the protons of vinyl group around 7.03, 4.94 and 4.62 ppm dramatically disappeared. Meanwhile, a new peak at 10.46 ppm which assigned to the released phenolic hydroxyl group emerged from the concomitant reaction. About the mass spectra, a same peak of [M H] (265.1m/z) for the isolated reaction product of compound-2 and the synthesized compound-2 were also found respectively. Especially, the isolated product of compound-2 displayed a same absorption (Fig. S17) and fluorescence spectrum (Fig. S18) with the synthesized compound-2. All these experimental facts support the proposed oxymercuration reaction and the breakage reaction of CN-vinyl with Hg2 þ .
(2.0 equiv., 50 μM), such as Li þ , Na þ , K þ , Mg2 þ , Ca2 þ , Al3 þ , Fe2 þ , Fe3 þ , Mn2 þ, Cu2 þ , Co2 þ , Ni2 þ , Pb2 þ , Zn2 þ , Sn2 þ , Ag þ , Cd2 þ , Ba2 þ , Cr3 þ , MeHg þ , Hg2 þ (50 μM) were investigated by fluorescent spectra in THF/water (2:8, v/v, PBS, pH ¼7.0) solution at λex ¼456 nm (Fig. 7). We can see that only Hg2 þ (50 μM) could cause a significant fluorescent decrease emission of CN-vinyl. Especially, MeHg þ , a specific organic interferent, has no obvious fluorescence response change to CN-vinyl due to its more weak electrophilic to vinyl group compared to Hg2 þ . Then, the interference of other metal ions was explored with three different sequences of addition of interfering ions and Hg2 þ . The process was described in detail in analytical procedure and no remarkable
+
+
2+
CN-vinyl Li Na K Mg 2+ 3+ 2+ 3+ 2+ Ca Al Fe Fe Mn 2+ 2+ 2+ 2+ 2+ Cu Co Ni Pb Zn 2+, + 2+ 2+ + Sn Ag Cd Ba Cr3 MeHgCl
450 400 350 300 250 200
Hg 2+
150 100 50 0 480
3.5. Selectivity of CN-vinyl to Hg2 þ over other metal ions Selectivity is a very important parameter for evaluating the performance of a chemosensor. Twenty-one kinds of metal ions
+
500
Fluorescence intensitity (a.u.)
868
530
580
630
Wavelength (nm)
Fig. 7. Fluorescent spectra of CN-vinyl (25 μM) in the presence of various metal ions (50 μM) in THF/water (2:8, v/v); All measurements were taken in a pH 7.0 PBS (50 mM) at 25 1C, Excitation wavelength was 456 nm.
c
b
a
d
Fig. 6. Partial 1H NMR spectra of CN-vinyl (25 mM) upon addition of Hg2 þ (2.0 equiv.) in DMSO-d6. (A) only CN-vinyl, (B) the isolated aggregates of compound-2 after CN-vinyl reacted with Hg2 þ in DMSO for 15 min, (C) the synthesized compound-2.
A. Wang et al. / Talanta 132 (2015) 864–870
500
CN-vinyl + Hg2+ + Mn+
869
CN-vinyl + Mn+
Fluorescence intensity (a.u.)
450 400 350 300 250 200 150 100 50 0 +
Li
+
Na
+
2+
2+
3+
2+
K Mg Ca Al Fe
3+
2+
2+
2+
2+
Fe Mn Cu Co Ni
2+
2+
Pb Zn Sn
2+,
+
2+
2+
Ag Cd Ba Cr3+ MeHg
+
Fig. 8. Fluorescence intensity effects from the reaction of CN-vinyl (25 μM) with Hg2 þ in the presence of other metal ions (50 μM) which was added after Hg2 þ . All measurements were taken in a pH 7.0 PBS (50 mM) at 25 1C, Excitation and emission were at 456 nm/537 nm respectively.
5. Conclusions
Table 1 Detection of Hg2 þ in real samples. Sample
Added (μM)
Detected (μM)
Recovery (%)
RSD (%)
Tap water
0 30 0 30 0 30
Not detected 30.82 Not detected 30.74 Not detected 30.38
– 102.73 – 102.49 – 101.27
– 2.91 – 3.29 – 1.91
Qinghe river Weiming lake
Table 2 Detection in certified reference material of standard solution of Hg2 þ . Sample
Diluted concentration (μM)
Detected (μM)
Recovery (%)
RSD (%)
Standard solution of Hg2 þ
25
25.32
101.28
2.80
In summary, we have designed and synthesized a “turn off” quantitative fluorescence probe CN-vinyl and confirmed by the standard spectroscopic analyses. CN-vinyl possesses unusual AIE characteristic, and interestingly, has remarkable sensing selectivity to Hg2 þ , which weaken with the increase of Hg2 þ and showed a good linear relation (R2 ¼ 0.9957) with Hg2 þ concentration in the range of 0 to 50 μM. There was no interference of coexist metal ions to Hg2 þ with this assay and it could be used to detect Hg2 þ with a low concentration limit of 37 nM. By means of NMR and MS analyses, the mechanism for Hg2 þ -mediated optical properties of CN-vinyl is depicted. It has a potential to be used to detect Hg2 þ ion in river, lake or tap water. The present study provides valuable information for designing fluorescent probes for Hg2 þ by taking advantage of the selective cleavage of vinyl group by Hg2 þ . Acknowledgments
changes were detected with the three assay ways in the presence of coexisting metal ions including organic mercury as shown in Fig. 8 and Figs. S19, S20. All these results showed that CN-vinyl has remarkable sensing selectivity to Hg2 þ , which made it suitable for practical Hg2 þ detection. 4. Analysis of Hg2 þ in real sample Three water samples were collected from Qinghe River in Beijing (sample 1), Weiming Lake in Beijing (sample 2) and tap water (sample 3). The results for the determination of Hg2 þ were shown in Table 1 and no Hg2 þ was detected in the three blank samples. When Hg2 þ was added to them, the recovery was determined three times by the standard addition method and in range of 100–103%. These results demonstrate that the proposed assay strategy is successful in the detection of Hg2 þ in real sample. In order to further demonstrate the accuracy of this method, the detection in certified reference material of standard solution of Hg2 þ was carried out according to the analytical procedure. The results are summarized in Table 2 and it shows good agreement between the expected and found values. All these data above indicate the proposed method can be used to analyze Hg2 þ accurately.
We thank the Science and Technology Innovation Foundation for the College Students of Beijing (No. 13220055), the National Natural Science Foundation of China (No. 20972015), and the Natural Science Foundation of Beijing (No. 2112026) for financial support. Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.talanta.2014.10.048. References [1] H.Y. Chang, T.M. Hsiung, Y.F. Huang, C.C. Huang, Environ. Sci. Technol. 45 (2011) 1534–1539. [2] H.H. Harris, I.J. Pickering, G.N. George, Science 301 (2003) 1203. [3] T. Agusa, T. Kunito, H. Iwata, I. Monirith, T.S. Tana, A. Subramanian, S. Tanabe, Environ. Pollut. 134 (2005) 79–86. [4] J.P. Bourdineaud, R. Rossignol, D. Brethes, Int. J. Biochem. Cell Biol. 45 (2013) 16–22. [5] C.B. Huang, H.R. Li, Y. Luo, L. Xu, Dalton Trans. 43 (2014) 8102–8108. [6] E.M. Nolan, S.J. Lippard, Chem. Rev. 108 (2008) 3443–3480. [7] I. Hoyle, R.D. Handy, Aquat. Toxicol. 72 (2005) 147–159. [8] N. Zhou, H. Chen, J. Li, L. Chen, Microchim. Acta 180 (2013) 493–499. [9] H.N. Kim, M.H. Lee, H.J. Kim, J.S. Kim, J. Yoon, Chem. Soc. Rev. 37 (2008) 1465–1472. [10] K.E. Lorber, Waste Manag. Res. 4 (1986) 3–13. [11] M.S. Hosseini, H. Hashemi-Moghaddam, Talanta 67 (2005) 555–559. [12] N.W. Khun, E. Liu, Electrochim. Acta 54 (2009) 2890–2898.
870
A. Wang et al. / Talanta 132 (2015) 864–870
[13] J. Manzoori, M. Sorouraddin, A.á.H Shabani, J. Anal. At. Spectrom. 13 (1998) 305–308. [14] A. Bernaus, X. Gaona, J.M. Esbrí, P. Higueras, G. Falkenberg, M. Valiente, Environ. Sci. Technol. 40 (2006) 4090–4095. [15] N.H. Bings, A. Bogaerts, J.A.C. Broekaert, Anal. Chem. 78 (2006) 3917–3946. [16] X. Xue, F. Wang, X. Liu, J. Am. Chem. Soc. 130 (2008) 3244–3245. [17] L.J. Fan, Y. Zhang, W.E. Jones, Macromolecules 38 (2005) 2844–2849. [18] C.C. Huang, H.T. Chang, Anal. Chem. 78 (2006) 8332–8338. [19] J.S. Lee, M.S. Han, C.A. Mirkin, Angew. Chem. 46 (2007) 4093–4096. [20] P. Li, H. Liu, L. Yang, J. Liu, Talanta 106 (2013) 381–387. [21] J. Luo, Z. Xie, J.W.Y. Lam, L. Cheng, B.Z. Tang, H. Chen, C. Qiu, H.S. Kwok, X. Zhan, Y. Liu, D. Zhu, Chem. Commun. (2001) 1740–1741. [22] R. Deans, J. Kim, M.R. Machacek, T.M. Swager, J. Am. Chem. Soc. 122 (2000) 8565–8566. [23] B.K. An, S.K. Kwon, S.-D. Jung, S.Y. Park, J. Am. Chem. Soc. 124 (2002) 14410–14415. [24] Z. Xie, B. Yang, G. Cheng, L. Liu, F. He, F. Shen, Y. Ma, S. Liu, Chem. Mater. 17 (2005) 1287–1289.
[25] S. Kim, Q. Zheng, G.S. He, D.J. Bharali, H.E. Pudavar, A. Baev, P.N. Prasad, Adv. Funct. Mater. 16 (2006) 2317–2323. [26] Y. Liu, X. Tao, F. Wang, J. Shi, J. Sun, W. Yu, Y. Ren, D. Zou, M. Jiang, J. Phys. Chem. C 111 (2007) 6544–6549. [27] H. Tong, Y. Dong, Y. Hong, M. Häussler, J.W. Lam, H.H.-Y. Sung, X. Yu, J. Sun, I.D. Williams, H.S. Kwok, J. Phys. Chem. C 111 (2007) 2287–2294. [28] Y. Liu, X. Tao, F. Wang, X. Dang, D. Zou, Y. Ren, M. Jiang, J. Phys. Chem. C 112 (2008) 3975–3981. [29] M. Wang, D. Zhang, G. Zhang, D. Zhu, Chem. Commun. (2008) 4469–4471. [30] B. Zhu, W. Wang, L. Liu, H. Jiang, B. Du, Q. Wei, Sens. Actuators B 191 (2014) 605–611. [31] S. Manohar, S.I. Khan, S.K. Kandi, K. Raj, G. Sun, X. Yang, A.D. Calderon Molina, N. Ni, B. Wang, D.S. Rawat, Bioorg. Med. Chem. Lett. 23 (2013) 112–116. [32] U.G. Lalloo, R. Naidoo, A. Ambaram, Curr. Opin. Pulm. Med. 12 (2006) 179–185. [33] J. Jiang, W. Liu, J. Cheng, L. Yang, H. Jiang, D. Bai, W. Liu, Chem. Commun. 48 (2012) 8371–8373.
Contents lists available at ScienceDirect
Talanta journal homepage: www.elsevier.com/locate/talanta
A highly selective and sensitive fluorescent probe for quantitative detection of Hg2 þ based on aggregation-induced emission features Aizhi Wang, Yunxu Yang n, Feifei Yu, Lingwei Xue, Biwei Hu, Weiping Fan, Yajun Dong Department of Chemistry and Chemical Engineering, School of Chemistry and Biological Engineering, University of Science and Technology Beijing, Beijing 100083, China
art ic l e i nf o
a b s t r a c t
Article history: Received 31 July 2014 Received in revised form 19 October 2014 Accepted 22 October 2014 Available online 31 October 2014
A π-conjugated cyanostilbene derivative of (Z)-2-(4-nitrophenyl)-3-(4-(vinyloxy)phenyl)acrylonitrile (CN-vinyl) had been designed, synthesized and confirmed by the standard spectroscopic analyses. CN-vinyl possesses an unusual high emissive aggregation-induced emission (AIE) feature in a tetrahydrofuran/water mixture (2:8, v/v). The fluorescence intensity of CN-vinyl can be quenched linearly with the addition of Hg2 þ in a range of 0–50 μM with a correlation coefficient of R2 ¼0.9957. The detection limit of Hg2 þ is 37 nM. The mechanism for Hg2 þ -mediated optical properties of CN-vinyl is due to the selective cleavage of vinyl group by Hg2 þ . Accuracy of the proposed methodology was evaluated by means of the recovery study in real samples and the analyzing certified reference material of the standard solution of Hg2 þ . By this novel strategy, CN-vinyl can be used for quantitative detection of Hg2 þ as well as the presence of other physiological relevant metal ions. & 2014 Elsevier B.V. All rights reserved.
Keywords: Fluorescent probe Quantitative detection Mercury ion Aggregation-induced emission
1. Introduction Mercury, a class of heavy metal distributed throughout the environment by both natural processes and human activities, has long been identified as one of the most toxic heavy metals and a kind of persistent contaminant [1,2]. It can first be transformed into organic mercury after being absorbed from the environment by organisms, and then accumulated highly in trophic animals through the food chain [3,4]. According to recent reports, the toxicity of Hg2 þ can induce a variety of syndromes and harmful effects [5], such as damage DNA, disable normal functions of liver and kidney, disrupt the immune system homeostasis [6–9] etc. Therefore, efficient monitoring the trace amount of Hg2 þ with highly selective and sensitive is remarkably important for human health. Conventional analytical techniques include spectrophotometry, atomic absorption spectrometry, inductively coupled plasma–mass spectroscopy (ICP–MS), cold-vapor atomic fluorescence spectrometry (CV-AFS), ultraviolet–visible spectrometry, and X-ray absorption spectroscopy [10–15]. Whereas, these methods require expensive intricate instrumentation and complicated sample preparation processes. Moreover, the complexity makes them unsuitable for using in on-site Hg2 þ analyses. Different from the traditional Hg2 þ analyses, fluorescence probe offers several advantages for Hg2 þ measurements because of its high sensitivity, lower detection limit,
n
Corresponding author. Tel.: þ 86-010-62333871 E-mail address: [email protected] (Y. Yang).
http://dx.doi.org/10.1016/j.talanta.2014.10.048 0039-9140/& 2014 Elsevier B.V. All rights reserved.
non-sample destructing and simple operation. In consequence, these unique characteristics make it favorable for both detection and imaging of Hg2 þ in biological samples [16]. Many Hg2 þ chemosensors with fluorescence response have been reported, such as, proteins conjugated polymer and diblock copolymer fluorescence sensors, biomolecules fluorescence sensors, foldamer-based fluorescence sensors, inorganic gold nanoparticles and CdS nanoparticles, and small organic molecule fluorescence probes [9,17–20]. However, luminescence of these conventional fluorophores often becomes weakened or quenched at high concentration or solid state due to the thermodynamically favorable process under the driving forces of intermolecular π–π stacking interaction of aggregation. The outcome of this effect is the sacrifice of the fluorescence emission efficiency and reduction of sensing performance. Fortunately, in 2001, Tang and co-workers first observed an intriguing phenomenon [21] that is the exact opposite of the aggregation-caused quenching (ACQ). Some practically nonluminescent in the solution state but become strongly emissive when aggregated or in the solid state due to suppression of nonradiative deactivation associated with restriction of intramolecular rotations (RIR). Then a large number of so called “aggregation-induced emission (AIE)” active molecules have since been developed, example of which including siloles, 1-cyano-trans-1, 2-bis-(40 -methylbiphenyl) ethylene (CN-MBE), 2, 5-diphenyl-1, 4-distyrylbenzene (DPDSB) derivatives, fluorenonearylamine derivatives, diphenyldibenzofulevne (DPDBF) derivatives, conjugated polymers, and so on [22–29]. Most of them can be employed as optical devices or bioprobes.
A. Wang et al. / Talanta 132 (2015) 864–870
865
Infrared spectra were recorded on a Shimadzu IR-8400S spectrophotometer. 1H NMR and 13C NMR spectra were recorded at 400 MHz on a Varian Gemin-400 respectively. ESI-MS spectra were obtained on Bruker spectrometers. All absorption and fluorescence spectra in this work were recorded in Pgeneral TU-1901 and Hitachi F-4500 fluorescence spectrometers. All pH values were measured with a Model pHS-3C pH meter (Shanghai, China). Analytical TLC was carried out on silica gel plates (HSGF254, 0.2 mm, Yantai Chemical Industry Research Institute). Melting points were measured with RD-II melting point apparatus (uncorrected, Tainjin Xintianguang Instrument Company).
Scheme 1. Synthesis of CN-vinyl.
Scheme 2. The reaction mechanism of CN-vinyl with Hg2 þ .
Zhu et al. have demonstrated that the vinyl group is useful to design Hg2 þ probe [30]. Inspired by their works, a novel fluorescence turn-off probe CN-vinyl ((Z)-2-(4-nitrophenyl)-3-(4-(vinyloxy)phenyl) acrylonitrile) toward Hg2 þ was designed and synthesized (as shown in Scheme 1). CN-vinyl is a potential AIE cyanostilbene derivatives based on π-conjugated fluorophore, and the vinyl group as a recognition site with unique and high reactivity toward Hg2 þ . We hypothesized that the reaction of CN-vinyl with Hg2 þ would result in the cleavage of the vinyl group and the recognition mechanism is shown in Scheme 2. In addition, compound-2 (Z)-3-(4-hydroxyphenyl)-2-(4-nitr-ophenyl)acrylonitrile was also synthesized as a contrast of CN-vinyl and the synthetic route is shown in Fig. S1.
2. Experimental section
2.2. Synthesis 2.2.1. Synthesis of 4-(2-bromoethoxy)benzaldehyde (compound-3) A similar method was adopted to synthesize the titled compound according to literature [31]. To a solution of p-hydroxybenzaldehyde (1.22 g, 0.01 mol) and anhydrous K2CO3 (4.14 g, 0.03 mol) in 20 ml DMF, requisite linear chain aliphatic dibromoethane (9.35 g, 0.05 mol) was added and then stirred at 80 1C for 1 h. After the reaction was finished (controlled by TLC), the reaction mixture was cooled to 0 1C and quenched with 1 M HCl. The mixture was then extracted with CHCl3 and the organic layer was washed with water, dried over anhydrous Na2SO4, and then filtered out. Excess solvent was evaporated under reduced pressure and the resultant solid left was purified by column chromatography (EtOAc/petroleum ether¼1:5 as eluent) to afford compound-3 (1.63 g, 72% yield). mp 52–54 1C. IR (KBr) v: 1685, 1602, 1577, 1506, 1253, 1163, 831 cm 1. 1 H NMR (400 MHz, CDCl3) δ: 9.88 (s, 1H), 7.83 (d, J¼ 8.8 Hz, 2H), 7.00 (d, J¼ 8.8 Hz, 2H), 4.36 (t, J¼ 6.4 Hz, 2H), 3.65 (t, J¼6.4 Hz, 2H).
2.2.2. Synthesis of 4-(vinyloxy)benzaldehyde (compound-4) A similar method was adopted to synthesize the titled compound according to the literature [32]. To a solution of compound-3 (0.227 g, 0.001 mol) in DMSO (10 mL), t-BuOK (0.224 g, 0.002 mol) was added. The reaction mixture was stirred at 25 1C for 4 h, after which it was diluted with EtOAc (20 mL), washed with H2O (3 10 mL) and then brine (20 mL). The organic layer was dried, filtered, and concentrated. The crude product was purified by column chromatography (EtOAc/petroleum ether¼1:5 as eluent) to afford compound-4 as a colorless oil (0.082 g, 56% yield). IR (KBr) v: 1697, 1645, 1598, 1504, 1299, 1249, 1161, 958, 833 cm 1. 1H NMR (400 MHz, CDCl3) δ (ppm): 9.90 (s, 1H), 7.85 (d, J¼8.8 Hz, 2H), 7.09 (d, J¼ 8.8 Hz, 2H), 6.67 (dd, J¼ 6.0 Hz, 13.6 Hz, 1H), 4.92 (dd, J¼2.0 Hz, 13.6 Hz, 1H), 4.61 (dd, J¼ 1.6 Hz, 6.0 Hz, 1H).
2.1. Reagents and apparatus All reagents and solvents were commercially available and were used without further purification unless otherwise stated. The water used for investigating was distilled prior to use. 4-nitrobenzylcyanide and p-hydroxybenzaldehyde were purchased from Alfa Aesar. MeHgCl was purchased from Dr. Ehrenstorfer Germany. The certified reference material of the standard solution of Hg2 þ was purchased from General Research Institute for Nonferrous Metals, China and the certificate information is shown in Table S1. Stock solutions of metal ions (2.5 mM, Li þ , Na þ , K þ , Mg2 þ , Ca2 þ , Al3 þ , Fe2 þ , Fe3 þ , Mn2 þ , Cu2 þ , Co2 þ , Ni2 þ , Pb2 þ , Zn2 þ , Sn2 þ , Ag þ , Cd2 þ , Ba2 þ , Cr3 þ , MeHg þ , Hg2 þ ) were prepared in deionized water. Stock solution of CN-vinyl (2.5 mM) was prepared by dissolving 18.2 mg CN-vinyl in 25 ml THF. Phosphate buffer solutions (PBS) were prepared by combining proper amounts of NaH2PO4 and NaOH and adjusted by a pH meter. Purification of reaction products was carried out by flash chromatography using silica gel (300–400 mesh, Branch of Qingdao Haiyang Chemical Co., Ltd.).
2.2.3. Synthesis of (Z)-2-(4-nitrophenyl)-3-(4-(vinyloxy)phenyl) acrylonitrile (CN-vinyl) To a mixture of compound-4 (0.148 g, 0.001 mol) and 4-nitrobenzylcyanide (0.162 g, 0.001 mol) in absolute EtOH (5 mL), piperidine (0.255 g, 0.003 mol) was added with dropwise. After the reaction mixture was stirred at room temperature for 3 h, the obtained precipitated was filtered, washed with EtOH, dried and purified by column chromatography (CH2Cl2/petroleum¼1:2 as eluent), a yellow solid was obtained. (0.233 g, 79% yield). mp 175–177 1C. IR (KBr) v: 2212, 1643, 1596, 1579, 1508, 1336, 1257, 1180, 848 cm 1. 1H NMR (400 MHz, DMSO-d) δ (ppm): 8.34 (d, J¼8.8 Hz, 2H), 8.28 (s, 1H), 8.06–8.00 (m, 4H), 7.27 (d, J¼8.8 Hz, 2H), 7.03 (dd, J¼6.0 Hz, 13.6 Hz, 1H), 4.89 (dd, J¼1.6 Hz, 13.6 Hz, 1H), 4.62 (dd, J¼ 1.6 Hz, 6.0 Hz, 1H). 13 C NMR (DMSO) δ (ppm): 158.9, 147.6, 147.3, 146.06, 140.7, 132.5, 132.3, 130.6, 128.3, 127.2, 124.8, 118.0, 117.1, 116.7, 106.7, 97.4, 97.3. ESIMS (m/z): calcd for: C17H12N203 [MþCl] 327.05, found 327.1. Anal. calcd for: C17H12N2O3, C, 69.86; H, 4.14; N, 9.58; found: C, 70.55; H, 4.07; N, 9.56.
A. Wang et al. / Talanta 132 (2015) 864–870
To characterize the AIE property of CN-vinyl, 100 μL stock solution of CN-vinyl (25 mM) was added to 10 mL THF/water (10:0, 4:6, 3:7, 2:8, 1:9, v/v, containing 50 mM NaH2PO4–NaOH). As shown in Fig. S10, the effect of water volume fraction on the pH was measured in THF/water containing 50 mM PBS. When THF and PBS was mixed well, the acidity of the solution decreased slightly (pH¼7.21–7.94) compared with PBS (pH ¼7.0). It is probably due to the restricting ionization of acid induced by THF. Then, the mixed solution was incubated at room temperature for 15 min and was transferred to a 1 cm quartz cell for absorption spectra and fluorescence spectra. In a standard detection method of Hg2 þ , the test solution was prepared by placing 100 μL stock solution of CN-vinyl and 1.9 ml THF into a test tube, diluted to 10 mL with 50 mM PBS at pH ¼7.0, added appropriate amount of Hg2 þ ions, incubate at room temperature for 15 min, and then record the fluorescence spectra. To evaluate any possible facts which may affect the response of the fluorescence intensity caused by interfering ions, three sequences of addition were attempted. (1) The interfering metal ions were added to the test solution, and then Hg2 þ was added to explore whether the reaction of CN-vinyl with Hg2 þ ions was affected by the interfering metal ions; (2) Hg2 þ ions were added to the test tube ahead, and then other interfering metal ions were added to explore whether the fluorescence intensity of reaction product was effected by interfering metal ions; (3) Hg2 þ ions and interfering metal ions were added to the test tubes at the same time to simulate the application in real sample analysis. All of them were allowed to incubate at room temperature for 15 min following to record the fluorescence spectra. For application in real sample analysis, the concentration of Hg2 þ in real samples was detected by the standard addition method. HgCl2 (4.1 mg) was added to 500 ml volumetric flask and diluted to metered volume with three real water samples which contain 50 mM PBS at pH ¼ 7.0 respectively, and gave 30 μM real sample solutions of Hg2 þ . Moreover, the certified standard solution of Hg2 þ (2.5 ml) was transferred to a 500 ml volumetric flask and diluted with PBS (50 mM, pH ¼ 7.0) to the metered volume, and give a 25 μM standard solution of Hg2 þ . Subsequently, 100 μL stock solutions of CN-vinyl and 1.9 ml THF were placed into test tubes and diluted to 10 mL with the three real samples and the standard solution of Hg2 þ respectively, and gave the test solutions. Finally, the fluorescence emission spectra were recorded. All fluorescence spectra were recorded in the range from 480 nm to 650 nm using a 456 nm excitation wavelength. The excitation and emission bandwidths were set to 5 nm and 10 nm respectively.
3. Results and discussion 3.1. Aggregation-induced emission characteristics of CN-vinyl We first studied the optical properties of CN-vinyl and compound-2. As shown in Fig. S11, both the absorption spectra of CNvinyl and compound-2 in THF solution exhibited three absorption bands at 245.0, 291.5, 365.5 nm and 249, 293.5, 368 nm respectively. Although CN-vinyl has a longer π-conjugated unit, it showed a blueshift spectrum due to a more twisted conformation caused by the cyano and vinyl group. This process had been supported by the theoretical calculations which was performed at B3LYP/6–31G (d) basis in the Gaussian 09 package. The optimized molecular structures and HOMO and LUMO energy levels of CN-vinyl and compound-2 are shown in Fig. S12. Obviously, both of them have
strong intramolecular charge transfer (ICT) effect from the electron cloud distribution. The calculated energy band gap for CN-vinyl is 3.456 eV, which is wider than that of compound-2 (3.392 eV). Hence, the results of theoretical studies are consistent with the absorption spectra. The characteristics of CN-vinyl were investigated in THF/water (from 10:0 to 1:9, v/v) phosphate buffer solution (PBS, 50 mM, pH ¼7.0) and the results are shown in Figs. 1 and 2. As expected, there was a structured absorption spectra and almost no fluorescence emission could be detected in dilute THF solution, suggesting that CN-vinyl is non-fluorescence when dispersed in its “solution” state. However, its fluorescence spectra in a THF/water mixture with a high water fraction (80%) had an intensive band at 537 nm, and showed a leveling-off in the ultraviolet region of the absorption spectra (commonly observed in nanoaggregates suspension). The fluorescence increased by 79 times compared with that in pure THF and a lower THF volume fraction was found to enhance the AIE fluorescence intensity. These results correlated well with the fact that more aggregates were formed in poorer solvents. On the other hand, the maximum peak in the absorption spectra of the nanoparticle suspension of CN-vinyl had a clearly red-shift (12 nm) compared with that of dilute solution (as shown in Fig. 2). This result implied that there is an extension of the effective conjugation length which was caused by the planarization of twist molecular in nanoparticles. The new shoulder band (the arrow) observed around 450 nm should be resulted from the
Fluorescence intensity (a.u.)
2.3. Analytical procedure
600
THF
500
60% Water 70% Water
400
80% Water
300
90% Water 200
100
0 480
530
580
630
Wavelength (nm)
600 500 Fluorescence intensity (a.u.)
866
400 300 200 100 0 0
20
40
60
80
100
Water fraction (%)
Fig. 1. (a) Fluorescent spectra of CN-vinyl (25 μM) in a THF/water mixture (excitation wavelength was 456 nm) (b) Effect of water volume fraction on the emission intensity of CN-vinyl (25 μM) in THF/water containing 50 mM PBS at pH 7.0. Inset show photographs of CN-vinyl (25 μM) in dilute THF solution and a THF/ water mixture with a high water fraction under a UV lamp (365 nm). Excitation and emission was at 456 nm/537 nm respectively.
A. Wang et al. / Talanta 132 (2015) 864–870
867
3.3. Fluorescence detection of Hg2 þ based on CN-vinyl
Fig. 2. Absorption spectra of CN-vinyl (25 μM) in a THF/water mixture.
450
CN-vinly
Fluorescence intensity (a.u.)
400 350
2+
CN-vinly + Hg
300 250
The fluorescence spectra of CN-vinyl upon titration with Hg2 þ was recorded in THF/water (2:8, v/v) buffered by 50 mM PBS at pH¼7.0 to investigated the quenching behavior of Hg2 þ ion. As shown in Fig. 4, CN-vinyl itself showed a very strong emission. However, the fluorescence of CN-vinyl (25 μM) at 537 nm was dramatically decreased upon the addition of Hg2 þ and followed a slight red-shift. Simultaneously, a perceived fluorescence color change of CN-vinyl was observed from yellow to colorless (Fig. 4, insert). It was plausible to suppose that a water-soluble species (should be compound-2) increased and the formation of aggregation of CN-vinyl decreased with the cleavage of the vinyl group upon the addition of Hg2 þ to CN-vinyl. The quenching of photoluminescence is observed at a lower concentration of Hg2 þ . The change of emission intensity became constant and caused 7-fold decrease when the addition of Hg2 þ added to 2.0 equiv. (50 μM). There was an approximately linear relationship (R2 ¼ 0.9957) between fluorescence intensity at 537 nm and Hg2 þ concentration in the range of 0 to 50 μM (Fig. 5). The detection limit was found to be 37 nM on the basis of the equation LOD ¼ 3δ/m (δ was the standard deviation of the blank solution and m is the absolute value of the slope between fluorescence intensity and Hg2 þ concentration) (Fig. S16). Above results demonstrated that our
200 150 100 50 0 1
2
3
4
5
6
7
8
9
10
11
12
13
pH
Fig. 3. Effect of pH on the emission intensity of probe CN-vinly (25 μM) at 537 nm in the absence and presence of 50 μM of Hg2 þ . All measurements were taken in PBS (50 mM, pH¼ 7.0) at25 1C. Excitation and emission were at 456 nm/537 nm respectively.
J-type aggregation. These date clearly indicated that CN-vinyl exhibits a significant feature of AIE.
The reaction conditions, such as pH, incubation time and buffer capacity of PBS, play an important role in the sensing process. The effects of pH value on the fluorescence intensity of CN-vinyl (25 μM), and the mixture of CN-vinyl (25 μM) with Hg2 þ (50 μM) in THF/ water (2:8, v/v) were investigated at first. As shown in Fig. 3, the fluorescence intensity of CN-vinyl decreased gradually with the rising of pH from 7.0 to 1.0 due to the formation of oxonium ion. However, when pH increased up to 10, there was almost no fluorescence because of the 1, 4-addition reaction of OH- to α, β-unsaturated cyano compound, which lead to destruction of the conjugate structure. As CN-vinyl displayed a strong fluorescence changes in the pH range of 6–9 upon the addition of Hg2 þ , and considering the application of samples in physiological or environmental, pH¼7.0 was thus chosen as the experiment condition. In addition, the results of timedependent fluorescence measurement of CN-vinyl (25 μM) and Hg2 þ (50 μM) in THF/water (2:8, v/v, PBS, pH¼7.0) is displayed in Fig. S13, of which would be quenched completely after 15 min. So, all the results of the assay were tested at the time of 15 min after the CN-vinyl was mixed with the Hg2 þ . Finally, the buffer capacity of PBS (pH¼7.0) was explored. As shown in Figs. S14 and 15, we could see that pH present a relatively stable value even if the concentration of Hg2 þ ions or other interfering metal ions reach its maximum (50 μM) in 50 mM PBS.
Fig. 4. Fluorescence spectra of CN-vinyl (25 μM) in the presence of different concentrations of Hg2 þ ion at a THF/water mixture (2:8, v/v); Inset: fluorescence color change of CN-vinyl (25 μM) in the absence and presence of Hg2 þ (2.0 equiv.). All measurements were taken in PBS (50 mM, pH 7.0) at 25 1C. Excitation wavelength was 456 nm.
450 400
Fluorescence intensity (a.u.)
3.2. Optimization of the analytical condition
350 300 y = -159.48x + 361.85 R² = 0.9957
250 200 150 100 50 0 0
0.5
1
1.5
2
2.5
3
3.5
equiv. of Hg2+
Fig. 5. The relationship between fluorescence intensity and Hg2 þ concentration. Plots of the fluorescence intensity obtained from the reaction of CN-vinyl (25 μM) with Hg2 þ (0–2.0 equiv.).All measurements were taken in PBS (50 mM, pH 7.0) at 25 1C. Excitation and emission were at 456 nm/537 nm respectively.
A. Wang et al. / Talanta 132 (2015) 864–870
proposed CN-vinyl could detect Hg2 þ in quantitative and with excellent sensitivity by the fluorescence spectrometry method. 3.4. Reaction mechanism It has been reported that Hg2 þ could selectively cleave the vinyl group by the oxymercuration reaction [33]. To verify our design principle of CN-vinyl for Hg2 þ , the reaction mechanism was studied by 1H NMR and mass spectra. Compound-2 was isolated from the reacting system to confirm that the breakage action of CN-vinyl with Hg2 þ had indeed produced after Hg2 þ was added to CN-vinyl. 1H NMR spectra of CN-vinyl, the isolated reaction product of compound-2 and the synthesized compound-2 were shown in Fig. 6A–C. Upon the addition of Hg2 þ , three protons of CN-vinyl corresponding to the protons of vinyl group around 7.03, 4.94 and 4.62 ppm dramatically disappeared. Meanwhile, a new peak at 10.46 ppm which assigned to the released phenolic hydroxyl group emerged from the concomitant reaction. About the mass spectra, a same peak of [M H] (265.1m/z) for the isolated reaction product of compound-2 and the synthesized compound-2 were also found respectively. Especially, the isolated product of compound-2 displayed a same absorption (Fig. S17) and fluorescence spectrum (Fig. S18) with the synthesized compound-2. All these experimental facts support the proposed oxymercuration reaction and the breakage reaction of CN-vinyl with Hg2 þ .
(2.0 equiv., 50 μM), such as Li þ , Na þ , K þ , Mg2 þ , Ca2 þ , Al3 þ , Fe2 þ , Fe3 þ , Mn2 þ, Cu2 þ , Co2 þ , Ni2 þ , Pb2 þ , Zn2 þ , Sn2 þ , Ag þ , Cd2 þ , Ba2 þ , Cr3 þ , MeHg þ , Hg2 þ (50 μM) were investigated by fluorescent spectra in THF/water (2:8, v/v, PBS, pH ¼7.0) solution at λex ¼456 nm (Fig. 7). We can see that only Hg2 þ (50 μM) could cause a significant fluorescent decrease emission of CN-vinyl. Especially, MeHg þ , a specific organic interferent, has no obvious fluorescence response change to CN-vinyl due to its more weak electrophilic to vinyl group compared to Hg2 þ . Then, the interference of other metal ions was explored with three different sequences of addition of interfering ions and Hg2 þ . The process was described in detail in analytical procedure and no remarkable
+
+
2+
CN-vinyl Li Na K Mg 2+ 3+ 2+ 3+ 2+ Ca Al Fe Fe Mn 2+ 2+ 2+ 2+ 2+ Cu Co Ni Pb Zn 2+, + 2+ 2+ + Sn Ag Cd Ba Cr3 MeHgCl
450 400 350 300 250 200
Hg 2+
150 100 50 0 480
3.5. Selectivity of CN-vinyl to Hg2 þ over other metal ions Selectivity is a very important parameter for evaluating the performance of a chemosensor. Twenty-one kinds of metal ions
+
500
Fluorescence intensitity (a.u.)
868
530
580
630
Wavelength (nm)
Fig. 7. Fluorescent spectra of CN-vinyl (25 μM) in the presence of various metal ions (50 μM) in THF/water (2:8, v/v); All measurements were taken in a pH 7.0 PBS (50 mM) at 25 1C, Excitation wavelength was 456 nm.
c
b
a
d
Fig. 6. Partial 1H NMR spectra of CN-vinyl (25 mM) upon addition of Hg2 þ (2.0 equiv.) in DMSO-d6. (A) only CN-vinyl, (B) the isolated aggregates of compound-2 after CN-vinyl reacted with Hg2 þ in DMSO for 15 min, (C) the synthesized compound-2.
A. Wang et al. / Talanta 132 (2015) 864–870
500
CN-vinyl + Hg2+ + Mn+
869
CN-vinyl + Mn+
Fluorescence intensity (a.u.)
450 400 350 300 250 200 150 100 50 0 +
Li
+
Na
+
2+
2+
3+
2+
K Mg Ca Al Fe
3+
2+
2+
2+
2+
Fe Mn Cu Co Ni
2+
2+
Pb Zn Sn
2+,
+
2+
2+
Ag Cd Ba Cr3+ MeHg
+
Fig. 8. Fluorescence intensity effects from the reaction of CN-vinyl (25 μM) with Hg2 þ in the presence of other metal ions (50 μM) which was added after Hg2 þ . All measurements were taken in a pH 7.0 PBS (50 mM) at 25 1C, Excitation and emission were at 456 nm/537 nm respectively.
5. Conclusions
Table 1 Detection of Hg2 þ in real samples. Sample
Added (μM)
Detected (μM)
Recovery (%)
RSD (%)
Tap water
0 30 0 30 0 30
Not detected 30.82 Not detected 30.74 Not detected 30.38
– 102.73 – 102.49 – 101.27
– 2.91 – 3.29 – 1.91
Qinghe river Weiming lake
Table 2 Detection in certified reference material of standard solution of Hg2 þ . Sample
Diluted concentration (μM)
Detected (μM)
Recovery (%)
RSD (%)
Standard solution of Hg2 þ
25
25.32
101.28
2.80
In summary, we have designed and synthesized a “turn off” quantitative fluorescence probe CN-vinyl and confirmed by the standard spectroscopic analyses. CN-vinyl possesses unusual AIE characteristic, and interestingly, has remarkable sensing selectivity to Hg2 þ , which weaken with the increase of Hg2 þ and showed a good linear relation (R2 ¼ 0.9957) with Hg2 þ concentration in the range of 0 to 50 μM. There was no interference of coexist metal ions to Hg2 þ with this assay and it could be used to detect Hg2 þ with a low concentration limit of 37 nM. By means of NMR and MS analyses, the mechanism for Hg2 þ -mediated optical properties of CN-vinyl is depicted. It has a potential to be used to detect Hg2 þ ion in river, lake or tap water. The present study provides valuable information for designing fluorescent probes for Hg2 þ by taking advantage of the selective cleavage of vinyl group by Hg2 þ . Acknowledgments
changes were detected with the three assay ways in the presence of coexisting metal ions including organic mercury as shown in Fig. 8 and Figs. S19, S20. All these results showed that CN-vinyl has remarkable sensing selectivity to Hg2 þ , which made it suitable for practical Hg2 þ detection. 4. Analysis of Hg2 þ in real sample Three water samples were collected from Qinghe River in Beijing (sample 1), Weiming Lake in Beijing (sample 2) and tap water (sample 3). The results for the determination of Hg2 þ were shown in Table 1 and no Hg2 þ was detected in the three blank samples. When Hg2 þ was added to them, the recovery was determined three times by the standard addition method and in range of 100–103%. These results demonstrate that the proposed assay strategy is successful in the detection of Hg2 þ in real sample. In order to further demonstrate the accuracy of this method, the detection in certified reference material of standard solution of Hg2 þ was carried out according to the analytical procedure. The results are summarized in Table 2 and it shows good agreement between the expected and found values. All these data above indicate the proposed method can be used to analyze Hg2 þ accurately.
We thank the Science and Technology Innovation Foundation for the College Students of Beijing (No. 13220055), the National Natural Science Foundation of China (No. 20972015), and the Natural Science Foundation of Beijing (No. 2112026) for financial support. Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.talanta.2014.10.048. References [1] H.Y. Chang, T.M. Hsiung, Y.F. Huang, C.C. Huang, Environ. Sci. Technol. 45 (2011) 1534–1539. [2] H.H. Harris, I.J. Pickering, G.N. George, Science 301 (2003) 1203. [3] T. Agusa, T. Kunito, H. Iwata, I. Monirith, T.S. Tana, A. Subramanian, S. Tanabe, Environ. Pollut. 134 (2005) 79–86. [4] J.P. Bourdineaud, R. Rossignol, D. Brethes, Int. J. Biochem. Cell Biol. 45 (2013) 16–22. [5] C.B. Huang, H.R. Li, Y. Luo, L. Xu, Dalton Trans. 43 (2014) 8102–8108. [6] E.M. Nolan, S.J. Lippard, Chem. Rev. 108 (2008) 3443–3480. [7] I. Hoyle, R.D. Handy, Aquat. Toxicol. 72 (2005) 147–159. [8] N. Zhou, H. Chen, J. Li, L. Chen, Microchim. Acta 180 (2013) 493–499. [9] H.N. Kim, M.H. Lee, H.J. Kim, J.S. Kim, J. Yoon, Chem. Soc. Rev. 37 (2008) 1465–1472. [10] K.E. Lorber, Waste Manag. Res. 4 (1986) 3–13. [11] M.S. Hosseini, H. Hashemi-Moghaddam, Talanta 67 (2005) 555–559. [12] N.W. Khun, E. Liu, Electrochim. Acta 54 (2009) 2890–2898.
870
A. Wang et al. / Talanta 132 (2015) 864–870
[13] J. Manzoori, M. Sorouraddin, A.á.H Shabani, J. Anal. At. Spectrom. 13 (1998) 305–308. [14] A. Bernaus, X. Gaona, J.M. Esbrí, P. Higueras, G. Falkenberg, M. Valiente, Environ. Sci. Technol. 40 (2006) 4090–4095. [15] N.H. Bings, A. Bogaerts, J.A.C. Broekaert, Anal. Chem. 78 (2006) 3917–3946. [16] X. Xue, F. Wang, X. Liu, J. Am. Chem. Soc. 130 (2008) 3244–3245. [17] L.J. Fan, Y. Zhang, W.E. Jones, Macromolecules 38 (2005) 2844–2849. [18] C.C. Huang, H.T. Chang, Anal. Chem. 78 (2006) 8332–8338. [19] J.S. Lee, M.S. Han, C.A. Mirkin, Angew. Chem. 46 (2007) 4093–4096. [20] P. Li, H. Liu, L. Yang, J. Liu, Talanta 106 (2013) 381–387. [21] J. Luo, Z. Xie, J.W.Y. Lam, L. Cheng, B.Z. Tang, H. Chen, C. Qiu, H.S. Kwok, X. Zhan, Y. Liu, D. Zhu, Chem. Commun. (2001) 1740–1741. [22] R. Deans, J. Kim, M.R. Machacek, T.M. Swager, J. Am. Chem. Soc. 122 (2000) 8565–8566. [23] B.K. An, S.K. Kwon, S.-D. Jung, S.Y. Park, J. Am. Chem. Soc. 124 (2002) 14410–14415. [24] Z. Xie, B. Yang, G. Cheng, L. Liu, F. He, F. Shen, Y. Ma, S. Liu, Chem. Mater. 17 (2005) 1287–1289.
[25] S. Kim, Q. Zheng, G.S. He, D.J. Bharali, H.E. Pudavar, A. Baev, P.N. Prasad, Adv. Funct. Mater. 16 (2006) 2317–2323. [26] Y. Liu, X. Tao, F. Wang, J. Shi, J. Sun, W. Yu, Y. Ren, D. Zou, M. Jiang, J. Phys. Chem. C 111 (2007) 6544–6549. [27] H. Tong, Y. Dong, Y. Hong, M. Häussler, J.W. Lam, H.H.-Y. Sung, X. Yu, J. Sun, I.D. Williams, H.S. Kwok, J. Phys. Chem. C 111 (2007) 2287–2294. [28] Y. Liu, X. Tao, F. Wang, X. Dang, D. Zou, Y. Ren, M. Jiang, J. Phys. Chem. C 112 (2008) 3975–3981. [29] M. Wang, D. Zhang, G. Zhang, D. Zhu, Chem. Commun. (2008) 4469–4471. [30] B. Zhu, W. Wang, L. Liu, H. Jiang, B. Du, Q. Wei, Sens. Actuators B 191 (2014) 605–611. [31] S. Manohar, S.I. Khan, S.K. Kandi, K. Raj, G. Sun, X. Yang, A.D. Calderon Molina, N. Ni, B. Wang, D.S. Rawat, Bioorg. Med. Chem. Lett. 23 (2013) 112–116. [32] U.G. Lalloo, R. Naidoo, A. Ambaram, Curr. Opin. Pulm. Med. 12 (2006) 179–185. [33] J. Jiang, W. Liu, J. Cheng, L. Yang, H. Jiang, D. Bai, W. Liu, Chem. Commun. 48 (2012) 8371–8373.
