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Reaction-based fluorescent probe for differential detection of cyanide and bisulfite in the aqueous media
Journal of Luminescence 215 (2019) 116620
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Reaction-based fluorescent probe for differential detection of cyanide and bisulfite in the aqueous media
T
Lubao Zhua, Jiaojiao Nieb, Qiang Lia, Jianshi Dud, Xuewen Fanc, Fuquan Baic, Qingbiao Yanga,*, Yaming Shanb,**, Yaoxian Lia a
College of Chemistry, Jilin University, Changchun, 130021, China National Engineering Laboratory of AIDS Vaccine, School of Life Sciences, Jilin University, Changchun, China c International Joint Research Laboratory of Nano-Micro Architecture Chemistry, Laboratory of Theoretical and Computational Chemistry, Institute of Theoretical Chemistry, Jilin University, Changchun, 130023, China d Jilin Provincial Key Laboratory of Lymphatic Surgery, China Japan Union Hospital, Jilin University, Changchun, 130031, China b
A B S T R A C T
A dual-channel fluorescent probe PHTP with excellent water solubility and detection limit was used. The detection selectivity of the probe with CN− or HSO3− was determined by the pH value of the solution. Probe reacted with HSO3− at the pH of solution was 7.4 and emit a fluorescence signal at 450 nm, but probe reacted with CN− at pH of 9.3 and an intense fluorescence emission with a peak at 507 nm was acquired, and mutual interference observed during the response process was scarce. In addition, other competitive biologically relevant species, including F─, Cl─, Br─, I─, NO3─, SO42─, SCN─, H2PO4─, CO32─, C2O42─, AcO─, N3─, NO2─, HCO3─, S2O32─, HS─, Cys, GSH, and Hcy, were also rare interfere. The reaction mechanism was investigated by direct HR-MS, 1H NMR and extensive theoretical calculation. Furthermore, the cytotoxicity of PHTP by the standard MTT assay has been examined, PHTP had good biocompatibility and low cytotoxicity, and the probe was applied in the detection of living cells.
1. Introduction As common industrial materials, cyanide and sulfur dioxide derivatives (sulfite and bisulfite) are widely employed in various fields. However, cyanide ions can quickly bind to ferric ions in cytochrome oxidase in vivo and consequently inhibit the activity of enzyme; this phenomenon restricts tissues to utilize oxygen, thereby leading to vomiting, convulsions, loss of consciousness, and eventually death [1]. The maximum content of cyanide in drinking water is 1.9 μM as stipulated by the World Health Organization. Similarly, bisulfite can also cause serious injuries to the stomach, intestines, and kidney due to excessive ingestion [2]. Therefore, developing highly sensitive, selective, and convenient methods for detection of cyanide and sulfur dioxide derivatives is of great importance [3,4]. Although many traditional instrument detection methods (electrochemistry, titration, and chromatography) have been developed to discriminatively determine cyanide and bisulfite, these methods possess some inconvenience in their practical application because of the expensive instrumentation required, the cumbersome testing process, and the long detection time [5]. Fluorescent probes were recently widely recognized as the optimal method for anions detection due to their sensitivity, excellent selectivity, low cost, and real-time detection [6,7].
*
According to previous reports, many types of fluorescent probes, such as coordination reaction, hydrogen-bonding interaction, and nucleophilic addition have also been explored [8–11]. Among these methods, nucleophilic addition of reaction probes has considerable advantages due to the unique chemical reaction that occurs between analytes and the probes during detection [12,13]. This reaction results in a stable adduct with an excellent fluorescent signal. Moreover, other analytes cannot react with the probe, except for the target analyte. In 2018, Zhao et al. reported a probe based on nucleophilic addition, and the signal was stable [14]. Thus, the reactive probes are widely employed in the research of anions with high selectivity and anti-interference, especially for cyanide and bisulfite [15,16]. Unfortunately, most of the probes can only detect cyanide or bisulfite [17–19], if cyanide and bisulfite co-exist in the same solution, then two different fluorescent probes must be used for separate detection, making this approach cumbersome and inefficient. Some reaction probes for dual-channel detection have been recently developed. In 2016, Chao et al. reported a fluorescent probe for detection of CN─, HSO3─, and extremely alkaline pH [20], but the organic solvent was massively employed in the detection process, and cannot be distinguished detected when analytes coexist in the same medium, the reaction activities between probe and analytes were simultaneously present. Therefore, the specificity of single reaction was
Corresponding author. College of Chemistry, Jilin University, Changchun, 130021, China Corresponding author. E-mail address: [email protected] (Q. Yang).
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https://doi.org/10.1016/j.jlumin.2019.116620 Received 6 March 2019; Received in revised form 9 July 2019; Accepted 11 July 2019 Available online 14 July 2019 0022-2313/ © 2019 Elsevier B.V. All rights reserved.
Journal of Luminescence 215 (2019) 116620
L. Zhu, et al.
Scheme 1. Reaction between probe PHTP and CN─ or HSO3─ under different pH.
Subsequently, the stability of probe and its applicable pH range were examined. The fluorescence intensity of PHTP (5 μM) at 507 nm in the absence of CN─ remained constant in the pH region from 4.0 to 9.3. After the addition of CN─, the fluorescence intensity was slowly increased from pH 7.0 and reached the maximum at pH 9.3. The addition of HSO3─ resulted in scarce fluorescence emission at 507 nm (Fig. 1c). By contrast, the fluorescence intensity of PHTP (10 μM) at 450 nm remained constant from pH 4.0 to 9.0 in the absence of HSO3─. Upon treatment with HSO3─, the fluorescence intensity rapidly increased in the pH region from 4.0 to 7.4, but gradually decreased in the pH region from 7.4 to 11.0. However, CN─ hardly reacted with the PHTP at pH 7.4 (Fig. 1d). Therefore, the results indicated that the selectivity in the reaction process was determined by pH. Moreover, PHTP can discriminatively detect CN─ and HSO3─ according to different UV–vis absorption spectra and fluorescence signal without mutual conflict.
unachievable in the same environment. In this case, a dual-channel fluorescent probe that can selectively react with the analytes and release the responding fluorescent signal under different conditions, such as temperature or pH, must be synthesized. Herein, a dual-channel reaction phenothiazine (PHTP) derivative fluorescent probe was designed for cyanide and bisulfite detection, based on the chemical reaction between the probe and analytes because the selectivity of the reaction procedure was determined by pH [21]. Moreover, probe PHTP for cyanide and bisulfite detection explored different fluorescence signals in the relative product with fluorescence emission channels (Scheme 1). Therefore, interference during the response process was scarce. In addition, the reaction mechanism between probe and analytes was developed by direct HR-MS, 1H NMR, and theoretical calculation to gain insight into the addition of cyanide and bisulfite position. Furthermore, PHTP was applied to the detection of biological cells attributed to the superior solubility of the probe in the water.
2.2. Investigation of spectral properties of PHTP The sensing ability of PHTP (5 μM) to detect CN─ in DMF/Tris-HCl (v/v = 1/99, pH = 9.3) buffer solution was investigated by UV–vis absorption spectroscopy at ambient temperature. As shown in Fig. 2a, PHTP itself demonstrated maximum absorption at 545 and 390 nm. With the increase in CN─ concentration, the maximum absorption peak at 545 and 390 nm gradually decreased, while another new absorption signal at 268 nm emerged with a clear isosbestic point at 320 nm. The absorption graphic stabilized after the addition of 14 eq CN─, and an evident color change from violet to colorless was detected by naked eyes (Fig. S1a), which implied that the nucleophilic attack of CN─ interrupted the π-conjugation of PHTP. Accordingly, the fluorescence spectrum of the probe PHTP to CN─ was investigated (Fig. 2b). The probe PHTP displayed a tenuous emission fluorescence band. Upon addition of increasing amount of CN─, a prominent enhancement of fluorescent intensity at 507 nm was acquired until the concentration of the probe was up to 14 eq. Consequently, the fluorescence color of the solutions changed from non-emissive to green, which was clearly captured by the naked eye under the illumination of a 365 nm UV lamp (Fig. S1b). This change indicated that the nucleophilic attack of CN─ to the indolium salt broke up the intramolecular ICT processes. The reaction time of PHTP toward CN─ were also investigated. The fluorescent intensity achieved the maximum within 13min (Fig. S2). A good linear relationship (R2 = 0.99) was observed with increasing cyanide concentration. In addition, the detection limit was 9.8 × 10 −8 M based
2. Results and discussion 2.1. Preliminary experiment An initial experiment has been conducted to investigate the spectrum response of PHTP (10 μM) for CN─ (DMF/Tris-HCl buffer, v/ v = 1/99, 10 mM, pH = 9.3) and HSO3─ (DMF/Tris-HCl buffer, v/ v = 1/99, 10 mM, pH = 7.4) in aqueous solution. All tests were performed at room temperature. As shown in Fig. 1a, the probe PHTP (10 μM) showed an intense absorption peak at 545 nm and a shoulder at 390 nm regardless of pH 7.4 or 9.3. Upon addition of excessive CN─, both the absorption bands decreased, showing a grown band at 268 nm accompanied with an evident violet to colorless. By contrast, when PHTP was treated with HSO3─, PHTP exhibited two grown absorption bands at 258 and 290 nm with decreasing of the original absorption peak at 545 and 390 nm, respectively, with a remarkable color change from violet to colorless. Thus, the absorption spectra displayed specific responses toward CN─ and HSO3─. Then, a simple test indicated that the probe PHTP was almost nonfluorescent. After the addition of excessive amounts of CN─, the fluorescent emission profiles stabilized and centered at 507 nm. By contrast, an intense fluorescence emission with a peak at 450 nm was observed after the addition of HSO3─ (Fig. 1b). The detection of CN─ and HSO3─ was attributed to the nucleophilic addition to the indolium group, in which the pH played a crucial effect. 2
Journal of Luminescence 215 (2019) 116620
L. Zhu, et al.
Fig. 1. (a) UV–vis absorption spectra and (b) fluorescence spectra response of PHTP (10 μM) for CN─ (DMF/Tris-HCl, pH = 9.3, λex = 268 nm) and HSO3─ (DMF/ Tris-HCl, pH = 7.4, λex = 258 nm) in aqueous solution. (c) Fluorescence intensity of probe PHTP (5 μM) in the absence CN─, presence CN─ and presence HSO3─ in DMF/Tris-HCl solution with different pH values. (d) Fluorescence intensity of probe PHTP (10 μM) in the absence HSO3─, presence HSO3─ and presence CN─ in DMF/ Tris-HCl solution with different pH values.
(Fig. 3b). The free probe PHTP exhibited very weak fluorescence emission at 450 nm. However, the addition of HSO3─ (0–24 μM) resulted in an increscent fluorescence peak in emission at 450 nm with a fluorescence color change from non-emissive to light blue under the illumination of 365 nm UV lamp (Fig. S3b). The reaction time of PHTP toward HSO3─ were also investigated. The fluorescent intensity reached the maximum within 1min. Moreover, a good linear relationship (R2 = 0.99) with the concentration of bisulfite was clearly observed, and the limits of detection were calculated to be 3.0 × 10 −8 M based on 3 σ/k.
on 3 σ/k. Similarly, the ability of probe PHTP (10 μM) to detect bisulfite in buffer solution (DMF/Tris-HCl, v/v = 1/99, pH = 7.4) at ambient temperature was also investigated. Upon the increasing of HSO3─, the absorption peak of PHTP at 545 and 390 nm was gradually decreased and a new broad band from around 258 to 290 nm was increased with a distinctly isosbestic point at 320 nm (Fig. 3a). The absorption curve stabilized and the color changed from violet to colorless (Fig. S3a), as seen by the naked eye after the addition of 2.4 eq HSO3─. Subsequently, the fluorescence responses of PHTP toward HSO3─ were investigated
Fig. 2. (a) UV–Vis spectra of PHTP (5 μM) in the presence of increased concentration of CN─ (0–14 eq) in DMF/Tris-HCl solution (v/v=1/99, pH=9.3). (b) The corresponding fluorescence spectra of PHTP, insert: fluorescent intensity ratio (F/F0) of PHTP with addition of CN─. 3
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Fig. 3. (a) The UV–Vis spectra of PHTP (10 μM) in the presence of increased concentration of HSO3─ (0-2.4 eq) in DMF/Tris-HCl solution (v/v=1/99, pH=7.4). (b) The corresponding fluorescence spectra of PHTP, insert: fluorescent intensity ratio (F/F0) of PHTP with addition of HSO3─.
[23] was used for the geometry optimization and absorption properties were simulated by Def2-TZVP [24]. Geometry optimization and vibrational frequency calculations for the singlet ground states were performed by the restricted density functional theory (RDFT). All the geometries were proved to be the stationary points. Time-dependent DFT (TDDFT) was used to simulate the absorption properties with considerations of the solvent effect of water by the integral equation formalism polarizable continuum model (IEFPCM). Table 1 presents the details of the absorption properties. The lowest absorption peak of CN─ was classified as a transition from HOMO to LUMO with a mixed LLCT/ILCT characters. The electron cloud mainly transferred from the left of phenothiazine to the right part and ethylene. The strongest absorption peak resulted from the transition from HOMO2 to LUMO with a mixed ILCT/LLCT character because of the major location of the orbitals on the right part of phenothiazine and ethylene. The similar proportion of the transition from HOMO to LUMO+4 mainly resulted from the right part of phenothiazine and ethylene to the left of phenothiazine. Fig. 6 shows that the energy gap between HOMO and LUMO of CN─ was 5.83 eV. The strongest absorption peak of HSO3─ was classified as a mixed transition from HOMO or HOMO-1 to LUMO, LUMO+2 or LUMO+3. The main characters resulted from the location of orbitals on the left of phenothiazine and transition from phenothiazine to benzene and N-contained five-membered ring. As shown in Fig. 6, the energy gap between HOMO and LUMO of HSO3─ was 6.24 eV, which was larger than of CN─. Hence, the emission wavelength of HSO3─ may be blue-shifted with that of CN─.
2.3. Selectivity and interference The selectivity of PHTP toward CN─ and HSO3─ over other competitive biologically relevant species, including F─, Cl─, Br─, I─, NO3─, SO42─, SCN─, H2PO4─, CO32─, C2O42─, AcO─, N3─, NO2─, HCO3─, S2O32─, HS─, Cys, GSH, and Hcy, was investigated by evaluating the fluorescence signal in buffer solution. As shown in Fig. 4a, after the addition of CN─ into the solution (DMF/Tris-HCl, v/v = 1/99, pH = 9.3) of PHTP (5 μM), a large enhancement of the fluorescence intensity was observed in the emission at 507 nm. Under the different conditions (10 μM PHTP and the solution DMF/Tris-HCl, v/v = 1/99, pH = 7.4), the most efficient fluorescence enhancing effect at 450 nm was induced by HSO3─/SO32─, and HS─ (50 μM) also showed a slight enhancing effect (Fig. 4b). By contrast, other relevant species showed negligible fluorescence enhancement even at high concentrations. Therefore, the results indicated that the presence of other substances did not react with PHTP or the reaction rate was very low, showing that probe PHTP exhibits high selectivity for CN─ and HSO3─ over other tested analytes.
2.4. Study of the sensing mechanism The sensing mechanism of probe PHTP toward CN─ and HSO3─ was explored based on the 1H NMR and the HR-MS spectral, respectively. As shown in Fig. 5, after treated with HSO3─, most of the protons of PHTP show a clearly shifted to the upfield was observed. The proton signal of the vinyl group (a, b) shifted upfield from 7.02 ppm to 6.94 ppm–4.73 ppm and 4.89 ppm (a1, b1), which indicated that the vinyl group was interrupted and the Intramolecular conjugation was disappeared. By contrast, on account of the nucleophilic attack of CN─ toward C-2, vinyl group (a2, b2) still conjugate with phenothiazine and proton signal of the vinyl group only tiny shifted to the upfield (7.02 and 6.94 ppm–4.89 ppm and 4.73 ppm). Moreover, the methylene proton H (c) adjacent to iminium cation showed a distinct upfield-shift upon treatment with HSO3─ and CN─. These results suggested that the nucleophilic attack of analytes converted the N+ in the indolenium to N, and the electron-withdrawing character was weakened. In addition, the HR-MS spectrum of combinative products [M-CN + H]+ and [MHSO3+H]+ was clearly observed as shown in Figs. S4 and S5, respectively. Theoretical calculations have been used to ensure the additional sites of different ions on the detection probe and explore the photophysical properties of products. The hybrid functional M06–2X [22] was employed for all the calculations. The triple zeta basis set Def-TZVP
2.5. Cell toxicity of probe PHTP In order to achieve the application of PHTP in living cells, we first examined the cytotoxicity of PHTP by the standard MTT assay. As shown in Fig. S10, the cell viability maintained above 80% after HeLa cells were incubated with different concentrations of PHTP (1–50 μM) for 24 h, suggesting that PHTP had good biocompatibility and low cytotoxicity. 2.6. Imaging of living cells The ability of PHTP to trace HSO3─ and CN─ in living cells was evaluated through the confocal fluorescence imaging (CLSM) in HeLa cells. As shown in Fig. 7a, the live HeLa cells were cultured with PHTP (10 μM) solution for 4 h at 37 °C, and the cells were almost nonfluorescent. However, after the cells were further incubated with HSO3─ 4
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Fig. 4. (a) Fluorescence responses of PHTP (5 μM) to CN─ (70 μM) in the presence of some other biological molecular species (700 μM). (b) The fluorescence responses of PHTP (10 μM) to HSO3─ (24 μM) in the presence of some other biological molecular species (240 μM), sodium hydrosulfide (50 μM) and bisulfite derivatives (24 μM).
(24 μM, pH = 7.4) for another 1 h, a prominent bright blue fluorescence was observed (Fig. 7b). By contrast, HeLa cells were incubated with PHTP (5 μM) for 4 h and the observed fluorescence was scarce (Fig. 8a). Upon adding CN─ (70 μM, pH = 9.3) and culturing for 1 h, a strong bright green fluorescence was observed (Fig. 8b). The results indicated that the probe PHTP has excellent cell permeability and the cellular environment did not disturb the nucleophilic reaction between PHTP with HSO3─ or CN─. Therefore, probe PHTP was capable of monitoring HSO3─ and CN─ in living cells, and these advantages made the probe a potential sensor for CN─ detection in alkaline environment like small intestine.
cell viability maintained above 80% after incubated with different concentrations of PHTP for 24 h, the probe has been successfully applied in cyanide and bisulfite detection in HeLa cells. Furthermore, since dual-channel detection probes with advantages of convenience and affordable, the probe likely applied to three-channel probe in the future by changing the detection conditions. Conflicts of interest “There are no conflicts to declare”. Acknowledgements
3. Conclusions We acknowledge the financial support from the National Natural Science Foundation of China (No. 21174052), Jilin Province Science and Technology Research Plan (No. 20170204039GX), and the Natural Science Foundation of Jilin Province of China (Nos. 20160101311JC and 20170101105JC).
Overall, a dual-channel fluorescent probe PHTP was synthesized and utilized for cyanide and bisulfite detection in an aqueous solution, in which the chemical reaction between the PHTP and analytes could express distinct emission signal to the corresponding product at different pH values. In addition, the probe could quantitatively determine cyanide and bisulfite with fast response and low limits of detection (for cyanide, LOD = 9.8 × 10 -8 M; for bisulfite, LOD = 3.0 × 10 -8 M). The reactive addition site on the probe PHTP was different, probe nucleophilic attack of CN− toward C-2 and HSO3− toward C-4. Moreover. The
Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jlumin.2019.116620. 5
Journal of Luminescence 215 (2019) 116620
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Fig. 5. 1H NMR spectra of PHTP and combinative product (PHTP + bisulfite and PHTP + cyanide).
Table 1 Electron transition configurations, oscillator strength (f), and assignment for the main absorption band of two products called CN─ and HSO3─. H means HOMO and L was LUMO. Name −
CN
HSO3-
State
Wav./Ene. (nm/eV)
Exp. (nm/eV)
S1 S4
346/3.58 268/4.63
268/4.63
S1 S3
331/3.74 277/4.47
290/4.28 268/4.63
Major Contri.
Osc. Strength
Assignment
H→L (84.4%) H-2→L (30.3%) H→L+4 (26.1%) H→L (76.2%) H→L+2 (42.4%) H-1→L (15.9%) H-1→L+3 (14.8%)
0.2133 0.6020
LLCT/ILCT ILCT/LLCT LLCT/ILCT ILCT LLCT/ILCT LLCT ILCT/LLCT
0.0522 0.5165
Fig. 6. Calculated energy levels, energy gaps (eV), and compositions (with different colors) of the selected molecular orbitals for CN─ (a) and HSO3─ (b). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) 6
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Fig. 7. Confocal fluorescence imaging of (a) living HeLa cells incubated with PHTP (10 μM), (b) 10 μM PHTP loaded HeLa cells further incubated with 24 μM of HSO3─ for another 1 h (Left: fluorescence image; middle: bright field image; right: merged image.)
Fig. 8. Confocal fluorescence imaging of (a) living HeLa cells incubated with probe (5 μM), (b) 5 μM PHTP loaded HeLa cells further incubated with 70 μM of CN─ for another 1 h (Left: fluorescence image; middle: bright field image; right: merged image.)
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Reaction-based fluorescent probe for differential detection of cyanide and bisulfite in the aqueous media
T
Lubao Zhua, Jiaojiao Nieb, Qiang Lia, Jianshi Dud, Xuewen Fanc, Fuquan Baic, Qingbiao Yanga,*, Yaming Shanb,**, Yaoxian Lia a
College of Chemistry, Jilin University, Changchun, 130021, China National Engineering Laboratory of AIDS Vaccine, School of Life Sciences, Jilin University, Changchun, China c International Joint Research Laboratory of Nano-Micro Architecture Chemistry, Laboratory of Theoretical and Computational Chemistry, Institute of Theoretical Chemistry, Jilin University, Changchun, 130023, China d Jilin Provincial Key Laboratory of Lymphatic Surgery, China Japan Union Hospital, Jilin University, Changchun, 130031, China b
A B S T R A C T
A dual-channel fluorescent probe PHTP with excellent water solubility and detection limit was used. The detection selectivity of the probe with CN− or HSO3− was determined by the pH value of the solution. Probe reacted with HSO3− at the pH of solution was 7.4 and emit a fluorescence signal at 450 nm, but probe reacted with CN− at pH of 9.3 and an intense fluorescence emission with a peak at 507 nm was acquired, and mutual interference observed during the response process was scarce. In addition, other competitive biologically relevant species, including F─, Cl─, Br─, I─, NO3─, SO42─, SCN─, H2PO4─, CO32─, C2O42─, AcO─, N3─, NO2─, HCO3─, S2O32─, HS─, Cys, GSH, and Hcy, were also rare interfere. The reaction mechanism was investigated by direct HR-MS, 1H NMR and extensive theoretical calculation. Furthermore, the cytotoxicity of PHTP by the standard MTT assay has been examined, PHTP had good biocompatibility and low cytotoxicity, and the probe was applied in the detection of living cells.
1. Introduction As common industrial materials, cyanide and sulfur dioxide derivatives (sulfite and bisulfite) are widely employed in various fields. However, cyanide ions can quickly bind to ferric ions in cytochrome oxidase in vivo and consequently inhibit the activity of enzyme; this phenomenon restricts tissues to utilize oxygen, thereby leading to vomiting, convulsions, loss of consciousness, and eventually death [1]. The maximum content of cyanide in drinking water is 1.9 μM as stipulated by the World Health Organization. Similarly, bisulfite can also cause serious injuries to the stomach, intestines, and kidney due to excessive ingestion [2]. Therefore, developing highly sensitive, selective, and convenient methods for detection of cyanide and sulfur dioxide derivatives is of great importance [3,4]. Although many traditional instrument detection methods (electrochemistry, titration, and chromatography) have been developed to discriminatively determine cyanide and bisulfite, these methods possess some inconvenience in their practical application because of the expensive instrumentation required, the cumbersome testing process, and the long detection time [5]. Fluorescent probes were recently widely recognized as the optimal method for anions detection due to their sensitivity, excellent selectivity, low cost, and real-time detection [6,7].
*
According to previous reports, many types of fluorescent probes, such as coordination reaction, hydrogen-bonding interaction, and nucleophilic addition have also been explored [8–11]. Among these methods, nucleophilic addition of reaction probes has considerable advantages due to the unique chemical reaction that occurs between analytes and the probes during detection [12,13]. This reaction results in a stable adduct with an excellent fluorescent signal. Moreover, other analytes cannot react with the probe, except for the target analyte. In 2018, Zhao et al. reported a probe based on nucleophilic addition, and the signal was stable [14]. Thus, the reactive probes are widely employed in the research of anions with high selectivity and anti-interference, especially for cyanide and bisulfite [15,16]. Unfortunately, most of the probes can only detect cyanide or bisulfite [17–19], if cyanide and bisulfite co-exist in the same solution, then two different fluorescent probes must be used for separate detection, making this approach cumbersome and inefficient. Some reaction probes for dual-channel detection have been recently developed. In 2016, Chao et al. reported a fluorescent probe for detection of CN─, HSO3─, and extremely alkaline pH [20], but the organic solvent was massively employed in the detection process, and cannot be distinguished detected when analytes coexist in the same medium, the reaction activities between probe and analytes were simultaneously present. Therefore, the specificity of single reaction was
Corresponding author. College of Chemistry, Jilin University, Changchun, 130021, China Corresponding author. E-mail address: [email protected] (Q. Yang).
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https://doi.org/10.1016/j.jlumin.2019.116620 Received 6 March 2019; Received in revised form 9 July 2019; Accepted 11 July 2019 Available online 14 July 2019 0022-2313/ © 2019 Elsevier B.V. All rights reserved.
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Scheme 1. Reaction between probe PHTP and CN─ or HSO3─ under different pH.
Subsequently, the stability of probe and its applicable pH range were examined. The fluorescence intensity of PHTP (5 μM) at 507 nm in the absence of CN─ remained constant in the pH region from 4.0 to 9.3. After the addition of CN─, the fluorescence intensity was slowly increased from pH 7.0 and reached the maximum at pH 9.3. The addition of HSO3─ resulted in scarce fluorescence emission at 507 nm (Fig. 1c). By contrast, the fluorescence intensity of PHTP (10 μM) at 450 nm remained constant from pH 4.0 to 9.0 in the absence of HSO3─. Upon treatment with HSO3─, the fluorescence intensity rapidly increased in the pH region from 4.0 to 7.4, but gradually decreased in the pH region from 7.4 to 11.0. However, CN─ hardly reacted with the PHTP at pH 7.4 (Fig. 1d). Therefore, the results indicated that the selectivity in the reaction process was determined by pH. Moreover, PHTP can discriminatively detect CN─ and HSO3─ according to different UV–vis absorption spectra and fluorescence signal without mutual conflict.
unachievable in the same environment. In this case, a dual-channel fluorescent probe that can selectively react with the analytes and release the responding fluorescent signal under different conditions, such as temperature or pH, must be synthesized. Herein, a dual-channel reaction phenothiazine (PHTP) derivative fluorescent probe was designed for cyanide and bisulfite detection, based on the chemical reaction between the probe and analytes because the selectivity of the reaction procedure was determined by pH [21]. Moreover, probe PHTP for cyanide and bisulfite detection explored different fluorescence signals in the relative product with fluorescence emission channels (Scheme 1). Therefore, interference during the response process was scarce. In addition, the reaction mechanism between probe and analytes was developed by direct HR-MS, 1H NMR, and theoretical calculation to gain insight into the addition of cyanide and bisulfite position. Furthermore, PHTP was applied to the detection of biological cells attributed to the superior solubility of the probe in the water.
2.2. Investigation of spectral properties of PHTP The sensing ability of PHTP (5 μM) to detect CN─ in DMF/Tris-HCl (v/v = 1/99, pH = 9.3) buffer solution was investigated by UV–vis absorption spectroscopy at ambient temperature. As shown in Fig. 2a, PHTP itself demonstrated maximum absorption at 545 and 390 nm. With the increase in CN─ concentration, the maximum absorption peak at 545 and 390 nm gradually decreased, while another new absorption signal at 268 nm emerged with a clear isosbestic point at 320 nm. The absorption graphic stabilized after the addition of 14 eq CN─, and an evident color change from violet to colorless was detected by naked eyes (Fig. S1a), which implied that the nucleophilic attack of CN─ interrupted the π-conjugation of PHTP. Accordingly, the fluorescence spectrum of the probe PHTP to CN─ was investigated (Fig. 2b). The probe PHTP displayed a tenuous emission fluorescence band. Upon addition of increasing amount of CN─, a prominent enhancement of fluorescent intensity at 507 nm was acquired until the concentration of the probe was up to 14 eq. Consequently, the fluorescence color of the solutions changed from non-emissive to green, which was clearly captured by the naked eye under the illumination of a 365 nm UV lamp (Fig. S1b). This change indicated that the nucleophilic attack of CN─ to the indolium salt broke up the intramolecular ICT processes. The reaction time of PHTP toward CN─ were also investigated. The fluorescent intensity achieved the maximum within 13min (Fig. S2). A good linear relationship (R2 = 0.99) was observed with increasing cyanide concentration. In addition, the detection limit was 9.8 × 10 −8 M based
2. Results and discussion 2.1. Preliminary experiment An initial experiment has been conducted to investigate the spectrum response of PHTP (10 μM) for CN─ (DMF/Tris-HCl buffer, v/ v = 1/99, 10 mM, pH = 9.3) and HSO3─ (DMF/Tris-HCl buffer, v/ v = 1/99, 10 mM, pH = 7.4) in aqueous solution. All tests were performed at room temperature. As shown in Fig. 1a, the probe PHTP (10 μM) showed an intense absorption peak at 545 nm and a shoulder at 390 nm regardless of pH 7.4 or 9.3. Upon addition of excessive CN─, both the absorption bands decreased, showing a grown band at 268 nm accompanied with an evident violet to colorless. By contrast, when PHTP was treated with HSO3─, PHTP exhibited two grown absorption bands at 258 and 290 nm with decreasing of the original absorption peak at 545 and 390 nm, respectively, with a remarkable color change from violet to colorless. Thus, the absorption spectra displayed specific responses toward CN─ and HSO3─. Then, a simple test indicated that the probe PHTP was almost nonfluorescent. After the addition of excessive amounts of CN─, the fluorescent emission profiles stabilized and centered at 507 nm. By contrast, an intense fluorescence emission with a peak at 450 nm was observed after the addition of HSO3─ (Fig. 1b). The detection of CN─ and HSO3─ was attributed to the nucleophilic addition to the indolium group, in which the pH played a crucial effect. 2
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Fig. 1. (a) UV–vis absorption spectra and (b) fluorescence spectra response of PHTP (10 μM) for CN─ (DMF/Tris-HCl, pH = 9.3, λex = 268 nm) and HSO3─ (DMF/ Tris-HCl, pH = 7.4, λex = 258 nm) in aqueous solution. (c) Fluorescence intensity of probe PHTP (5 μM) in the absence CN─, presence CN─ and presence HSO3─ in DMF/Tris-HCl solution with different pH values. (d) Fluorescence intensity of probe PHTP (10 μM) in the absence HSO3─, presence HSO3─ and presence CN─ in DMF/ Tris-HCl solution with different pH values.
(Fig. 3b). The free probe PHTP exhibited very weak fluorescence emission at 450 nm. However, the addition of HSO3─ (0–24 μM) resulted in an increscent fluorescence peak in emission at 450 nm with a fluorescence color change from non-emissive to light blue under the illumination of 365 nm UV lamp (Fig. S3b). The reaction time of PHTP toward HSO3─ were also investigated. The fluorescent intensity reached the maximum within 1min. Moreover, a good linear relationship (R2 = 0.99) with the concentration of bisulfite was clearly observed, and the limits of detection were calculated to be 3.0 × 10 −8 M based on 3 σ/k.
on 3 σ/k. Similarly, the ability of probe PHTP (10 μM) to detect bisulfite in buffer solution (DMF/Tris-HCl, v/v = 1/99, pH = 7.4) at ambient temperature was also investigated. Upon the increasing of HSO3─, the absorption peak of PHTP at 545 and 390 nm was gradually decreased and a new broad band from around 258 to 290 nm was increased with a distinctly isosbestic point at 320 nm (Fig. 3a). The absorption curve stabilized and the color changed from violet to colorless (Fig. S3a), as seen by the naked eye after the addition of 2.4 eq HSO3─. Subsequently, the fluorescence responses of PHTP toward HSO3─ were investigated
Fig. 2. (a) UV–Vis spectra of PHTP (5 μM) in the presence of increased concentration of CN─ (0–14 eq) in DMF/Tris-HCl solution (v/v=1/99, pH=9.3). (b) The corresponding fluorescence spectra of PHTP, insert: fluorescent intensity ratio (F/F0) of PHTP with addition of CN─. 3
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Fig. 3. (a) The UV–Vis spectra of PHTP (10 μM) in the presence of increased concentration of HSO3─ (0-2.4 eq) in DMF/Tris-HCl solution (v/v=1/99, pH=7.4). (b) The corresponding fluorescence spectra of PHTP, insert: fluorescent intensity ratio (F/F0) of PHTP with addition of HSO3─.
[23] was used for the geometry optimization and absorption properties were simulated by Def2-TZVP [24]. Geometry optimization and vibrational frequency calculations for the singlet ground states were performed by the restricted density functional theory (RDFT). All the geometries were proved to be the stationary points. Time-dependent DFT (TDDFT) was used to simulate the absorption properties with considerations of the solvent effect of water by the integral equation formalism polarizable continuum model (IEFPCM). Table 1 presents the details of the absorption properties. The lowest absorption peak of CN─ was classified as a transition from HOMO to LUMO with a mixed LLCT/ILCT characters. The electron cloud mainly transferred from the left of phenothiazine to the right part and ethylene. The strongest absorption peak resulted from the transition from HOMO2 to LUMO with a mixed ILCT/LLCT character because of the major location of the orbitals on the right part of phenothiazine and ethylene. The similar proportion of the transition from HOMO to LUMO+4 mainly resulted from the right part of phenothiazine and ethylene to the left of phenothiazine. Fig. 6 shows that the energy gap between HOMO and LUMO of CN─ was 5.83 eV. The strongest absorption peak of HSO3─ was classified as a mixed transition from HOMO or HOMO-1 to LUMO, LUMO+2 or LUMO+3. The main characters resulted from the location of orbitals on the left of phenothiazine and transition from phenothiazine to benzene and N-contained five-membered ring. As shown in Fig. 6, the energy gap between HOMO and LUMO of HSO3─ was 6.24 eV, which was larger than of CN─. Hence, the emission wavelength of HSO3─ may be blue-shifted with that of CN─.
2.3. Selectivity and interference The selectivity of PHTP toward CN─ and HSO3─ over other competitive biologically relevant species, including F─, Cl─, Br─, I─, NO3─, SO42─, SCN─, H2PO4─, CO32─, C2O42─, AcO─, N3─, NO2─, HCO3─, S2O32─, HS─, Cys, GSH, and Hcy, was investigated by evaluating the fluorescence signal in buffer solution. As shown in Fig. 4a, after the addition of CN─ into the solution (DMF/Tris-HCl, v/v = 1/99, pH = 9.3) of PHTP (5 μM), a large enhancement of the fluorescence intensity was observed in the emission at 507 nm. Under the different conditions (10 μM PHTP and the solution DMF/Tris-HCl, v/v = 1/99, pH = 7.4), the most efficient fluorescence enhancing effect at 450 nm was induced by HSO3─/SO32─, and HS─ (50 μM) also showed a slight enhancing effect (Fig. 4b). By contrast, other relevant species showed negligible fluorescence enhancement even at high concentrations. Therefore, the results indicated that the presence of other substances did not react with PHTP or the reaction rate was very low, showing that probe PHTP exhibits high selectivity for CN─ and HSO3─ over other tested analytes.
2.4. Study of the sensing mechanism The sensing mechanism of probe PHTP toward CN─ and HSO3─ was explored based on the 1H NMR and the HR-MS spectral, respectively. As shown in Fig. 5, after treated with HSO3─, most of the protons of PHTP show a clearly shifted to the upfield was observed. The proton signal of the vinyl group (a, b) shifted upfield from 7.02 ppm to 6.94 ppm–4.73 ppm and 4.89 ppm (a1, b1), which indicated that the vinyl group was interrupted and the Intramolecular conjugation was disappeared. By contrast, on account of the nucleophilic attack of CN─ toward C-2, vinyl group (a2, b2) still conjugate with phenothiazine and proton signal of the vinyl group only tiny shifted to the upfield (7.02 and 6.94 ppm–4.89 ppm and 4.73 ppm). Moreover, the methylene proton H (c) adjacent to iminium cation showed a distinct upfield-shift upon treatment with HSO3─ and CN─. These results suggested that the nucleophilic attack of analytes converted the N+ in the indolenium to N, and the electron-withdrawing character was weakened. In addition, the HR-MS spectrum of combinative products [M-CN + H]+ and [MHSO3+H]+ was clearly observed as shown in Figs. S4 and S5, respectively. Theoretical calculations have been used to ensure the additional sites of different ions on the detection probe and explore the photophysical properties of products. The hybrid functional M06–2X [22] was employed for all the calculations. The triple zeta basis set Def-TZVP
2.5. Cell toxicity of probe PHTP In order to achieve the application of PHTP in living cells, we first examined the cytotoxicity of PHTP by the standard MTT assay. As shown in Fig. S10, the cell viability maintained above 80% after HeLa cells were incubated with different concentrations of PHTP (1–50 μM) for 24 h, suggesting that PHTP had good biocompatibility and low cytotoxicity. 2.6. Imaging of living cells The ability of PHTP to trace HSO3─ and CN─ in living cells was evaluated through the confocal fluorescence imaging (CLSM) in HeLa cells. As shown in Fig. 7a, the live HeLa cells were cultured with PHTP (10 μM) solution for 4 h at 37 °C, and the cells were almost nonfluorescent. However, after the cells were further incubated with HSO3─ 4
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Fig. 4. (a) Fluorescence responses of PHTP (5 μM) to CN─ (70 μM) in the presence of some other biological molecular species (700 μM). (b) The fluorescence responses of PHTP (10 μM) to HSO3─ (24 μM) in the presence of some other biological molecular species (240 μM), sodium hydrosulfide (50 μM) and bisulfite derivatives (24 μM).
(24 μM, pH = 7.4) for another 1 h, a prominent bright blue fluorescence was observed (Fig. 7b). By contrast, HeLa cells were incubated with PHTP (5 μM) for 4 h and the observed fluorescence was scarce (Fig. 8a). Upon adding CN─ (70 μM, pH = 9.3) and culturing for 1 h, a strong bright green fluorescence was observed (Fig. 8b). The results indicated that the probe PHTP has excellent cell permeability and the cellular environment did not disturb the nucleophilic reaction between PHTP with HSO3─ or CN─. Therefore, probe PHTP was capable of monitoring HSO3─ and CN─ in living cells, and these advantages made the probe a potential sensor for CN─ detection in alkaline environment like small intestine.
cell viability maintained above 80% after incubated with different concentrations of PHTP for 24 h, the probe has been successfully applied in cyanide and bisulfite detection in HeLa cells. Furthermore, since dual-channel detection probes with advantages of convenience and affordable, the probe likely applied to three-channel probe in the future by changing the detection conditions. Conflicts of interest “There are no conflicts to declare”. Acknowledgements
3. Conclusions We acknowledge the financial support from the National Natural Science Foundation of China (No. 21174052), Jilin Province Science and Technology Research Plan (No. 20170204039GX), and the Natural Science Foundation of Jilin Province of China (Nos. 20160101311JC and 20170101105JC).
Overall, a dual-channel fluorescent probe PHTP was synthesized and utilized for cyanide and bisulfite detection in an aqueous solution, in which the chemical reaction between the PHTP and analytes could express distinct emission signal to the corresponding product at different pH values. In addition, the probe could quantitatively determine cyanide and bisulfite with fast response and low limits of detection (for cyanide, LOD = 9.8 × 10 -8 M; for bisulfite, LOD = 3.0 × 10 -8 M). The reactive addition site on the probe PHTP was different, probe nucleophilic attack of CN− toward C-2 and HSO3− toward C-4. Moreover. The
Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jlumin.2019.116620. 5
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Fig. 5. 1H NMR spectra of PHTP and combinative product (PHTP + bisulfite and PHTP + cyanide).
Table 1 Electron transition configurations, oscillator strength (f), and assignment for the main absorption band of two products called CN─ and HSO3─. H means HOMO and L was LUMO. Name −
CN
HSO3-
State
Wav./Ene. (nm/eV)
Exp. (nm/eV)
S1 S4
346/3.58 268/4.63
268/4.63
S1 S3
331/3.74 277/4.47
290/4.28 268/4.63
Major Contri.
Osc. Strength
Assignment
H→L (84.4%) H-2→L (30.3%) H→L+4 (26.1%) H→L (76.2%) H→L+2 (42.4%) H-1→L (15.9%) H-1→L+3 (14.8%)
0.2133 0.6020
LLCT/ILCT ILCT/LLCT LLCT/ILCT ILCT LLCT/ILCT LLCT ILCT/LLCT
0.0522 0.5165
Fig. 6. Calculated energy levels, energy gaps (eV), and compositions (with different colors) of the selected molecular orbitals for CN─ (a) and HSO3─ (b). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) 6
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Fig. 7. Confocal fluorescence imaging of (a) living HeLa cells incubated with PHTP (10 μM), (b) 10 μM PHTP loaded HeLa cells further incubated with 24 μM of HSO3─ for another 1 h (Left: fluorescence image; middle: bright field image; right: merged image.)
Fig. 8. Confocal fluorescence imaging of (a) living HeLa cells incubated with probe (5 μM), (b) 5 μM PHTP loaded HeLa cells further incubated with 70 μM of CN─ for another 1 h (Left: fluorescence image; middle: bright field image; right: merged image.)
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