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Dual-site fluorescent probe for multi-response detection of ClO− and H2O2 and bio-imaging
Analytica Chimica Acta xxx (xxxx) xxx
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Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca
Dual-site fluorescent probe for multi-response detection of ClO and H2O2 and bio-imaging Yuchao Du a, Bowei Wang a, b, c, **, Di Jin a, b, Mingrui Li a, Yang Li a, b, c, Xilong Yan a, b, c, Xueqin Zhou a, b, c, Ligong Chen a, b, c, * a b c
School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300350, PR China Collaborative Innovation Center of Chemical Science and Engineering, Tianjin, PR China Tianjin Engineering Research Center of Functional Fine Chemicals, Tianjin, PR China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
The first dual-site fluorescent probe (Geisha-1) for the simultaneous detection of ClO and H2O2 was developed. The probe presented three different fluorescence response modes toward ClO, H2O2 and ClO þ H2O2 respectively. The probe could achieve quantitative detection of ClO and H2O2 with both excellent sensitivity and high selectivity. Geisha-1 was successfully applied to detect intracellular and intra-tissue ClO and H2O2.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 September 2019 Received in revised form 2 December 2019 Accepted 19 December 2019 Available online xxx
Hypochlorite (ClO) and hydrogen peroxide (H2O2) commonly coexist in organism and are involved in the same physiological and pathological processes. So it is of great importance to develop fluorescent probes to detect both simultaneously. Herein, we reported the first dual-site fluorescent probe (Geisha1) for the quantitative detection of ClO and H2O2. This probe is constructed by chemically grafting N,Ndimethylthiocarbamate and borate to a fluorescence resonance energy transfer (FRET) platform. As a result, Geisha-1 not only presents three different responses to ClO, H2O2, and ClO þ H2O2 (the coexistence of ClO and H2O2) with high sensitivity and selectivity, but also exhibits low toxicity and cell membrane and tissue permeability, and it was further successfully applied to image ClO and H2O2 in living cells and tissues. Thus, Geisha-1 provides a promising application prospect in biological systems and an alternative strategy for the construction of dual-site fluorescent probes aiming at the multiresponse detection of other biologically relevant analytes. © 2019 Elsevier B.V. All rights reserved.
Keywords: Fluorescent probe Dual-site Hypochlorite Hydrogen peroxide Bio-imaging
* Corresponding author. School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300350, PR China. ** Corresponding author. School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300350, PR China. E-mail addresses: [email protected] (B. Wang), [email protected] (L. Chen).
1. Introduction Reactive oxygen species (ROS) is a class of free radicals or nonradical oxygenated molecules those are highly reactive to biomolecules [1]. ROS are normally generated along with oxygen
https://doi.org/10.1016/j.aca.2019.12.059 0003-2670/© 2019 Elsevier B.V. All rights reserved.
Please cite this article as: Y. Du et al., Dual-site fluorescent probe for multi-response detection of ClO and H2O2 and bio-imaging, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.12.059
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spectrometer with tetramethylsilane (TMS) as an internal standard. High-resolution mass spectra were recorded with a Bruker Daltonics microTOF-Q II instrument (ESI). All pH measurements were carried out with a Sartorius basic pH-meter PB-10. UVevis absorption spectra were obtained by using an L8 spectrophotometer. Fluorescence spectra were measured on a Hitachi F-2500 fluorescence spectrophotometer. Fluorescence quantum yields were determined by a FluoroMax-4 (Horiba Jobin Yvon) fluorometer equipped with an integrated sphere. For the fluorescence measurements, the excitation and emission slit widths were both 10 nm and the excitation wavelengths were 400/450 nm. Standard cell viability protocols (MTT assay) was taken on a spectra Max 190 microplate reader. The cell imaging was performed on a Leica TCS SP8 confocal laser scanning microscopy.
metabolism in the human immune system and plays an important role in cell signaling, migration and anti-inflammatory regulation [2,3]. As two important members of the ROS family, both hypochlorite (ClO) and hydrogen peroxide (H2O2) play a key regulatory role in normal cells [4e9]. However, excessive or misplaced ClO and H2O2 will cause oxidative stress and related diseases [10e14]. Therefore, monitoring of intracellular ClO and H2O2 is of great significance. Furthermore, it is meaningful to simultaneously detect ClO and H2O2, because they coexist in some cells and both synergistically regulate certain physiological processes or are involved in the same pathological processes. For example, in the immune system, H2O2 serves as a signal transducer, which activates macrophages to release ClO that can directly kill microorganisms [15e17]. In addition, increasing evidences have shown that both ClO and H2O2 are involved in neurodegenerative Alzheimer’s and Parkinson’s diseases, cardiovascular diseases and certain cancers [18e22]. However, distinguishing ClO and H2O2 is still challenging due to their similar chemical properties. Therefore, it is urgent to develop a method to simultaneously detect ClO and H2O2 with good selectivity, for the better understanding their complicated interrelationship and related physiological and pathological processes. Although colorimetric [23,24], electrochemical [25,26], and chromatographic [27,28] analytical methods, have been adopted to detect ClO or H2O2, the fluorescence analysis stood out to be the most eye-catching one due to its low cost, high sensitivity, noninvasive, and deep imaging ability [29e32]. To date, a number of probes toward ClO or H2O2 have been reported [33e37]. However, none of them can detect ClO and H2O2 synchronously. Simultaneous using of two corresponding probes seems to be a viable strategy, but it is inevitably subject to the cross-talk, stronger invasive effect, and different location and metabolism, making imaging more complicated and difficult to control. The dual-site probes proposed recently [38e43], which were equipped with two flexible recognition sites to overcome the above problems, are especially suitable for the simultaneous detection of two biomolecules. However, to the best of our knowledge, the dual-site probe capable of simultaneously imaging ClO and H2O2 has not been reported. Furthermore, the reported dual-site probes always just showed two responses toward different analytes respectively and lack of a third response to the coexisting analytes, which may cause the interference and distortion of the signal. Therefore, it is necessary to introduce a third response to the dual-site probe, namely two sites with triple responses, which is capable of differentiating ClO, H2O2, and ClO þ H2O2 (the coexistence of ClO and H2O2). Fluorescence resonance energy transfer (FRET) allows the probe to emit the fluorescence of acceptor fluorophore at the excitation wavelength of donor fluorophore, which makes it an effective tool to introduce a third response. In short, a coumarin-naphthalimide FRET platform was constructed and a dual-site fluorescent probe (Geisha-1) with three different responses to ClO, H2O2, and ClO þ H2O2 was developed.
Hela cells were used for cytotoxicity assay of probe Geisha-1 based on the standard cell viability protocols (MTT assay). Hela cells were grown in Dulbecco’s modified eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) in an atmosphere of 5% CO2 and 95% air at 37 C. Before the experiments, the cells were plated into a 96-well plate at a density of 5 105 cells per well and allowed to adhere for 24 h. Subsequently, the medium was replaced with fresh medium including 5, 10, 15, and 20 mM of Geisha-1. The cells that has not been treated with Geisha-1 were used as the blank control. After further incubation for 24 h, MTT solution (5 mg/ mL in PBS, 10 mM) was added to each well and incubation for another 4 h. Then the MTT solution was removed and 100 mL DMSO was added to dissolve the formazan crystals. After shaking for 10 min, the absorbance was measured at 570 nm using a microplate reader (spectra Max 190).
2. Experimental
2.4. Cell imaging
2.1. Materials and general methods
The Hela Cells were grown in DEME supplemented with 12% FBS in an atmosphere of 5% CO2 and 95% air at 37 C. The cells were plated on 35 mm glass bottom dishes and allowed to grow for 24 h. Before the experiments, cells were washed with PBS buffer. In each set of experiments, cells were incubated with Geisha-1 (10 mM) for 1.5 h at 37 C and then washed with PBS buffer 3 times. The cells were subsequently incubated with NaClO (200 mM), H2O2 (1 mM) and NaClO (200 mM) þ H2O2 (1 mM) for 30 min, 1 h and 1 h respectively. Cell imaging was carried out after washing the cells
All reagents and solvents were purchased from commercial sources and used without further purification unless otherwise noted. Deionized water was used throughout this work. Reactions were monitored by thin-layer chromatography (TLC) on silica gelprecoated glass plates. Silica gel (particle size 200e400 mesh) was used for column chromatography. 1H NMR and 13C NMR spectra were recorded with a Bruker Avance III 400 MHz
2.2. Synthesis of the probe Geisha-1 Compound 4 (1.64 g, 3.0 mmol) and triethylamine (0.90 g, 9.0 mmol) were dissolved in 30 mL anhydrous dichloromethane, and then a solution of N,N-dimethylaminothioformyl chloride (2.20 g, 18.0 mmol) in 3 mL anhydrous dichloromethane was added dropwise at 0 C. After stirring below 0 C for 90 min, the mixture was stirred at room temperature for 10 h. The solvent was removed by evaporation and the residue was purified by silica gel column chromatography (dichloromethane/methanol ¼ 100:1, v/v) to afford the desired Geisha-1 as a white solid (1.17 g, 60.8% yield). 1H NMR (400 MHz, CDCl3), d 9.12 (d, J ¼ 8.3 Hz, 1H), 8.96 (s, 1H), 8.83 (s, 1H), 8.60 (dd, J ¼ 14.0, 7.2 Hz, 2H), 8.29 (d, J ¼ 7.2 Hz, 1H), 7.77 (t, J ¼ 7.8 Hz, 1H), 7.65 (d, J ¼ 8.9 Hz, 1H), 7.11 (s, 2H), 4.53 (t, J ¼ 5.4 Hz, 2H), 3.88 (d, J ¼ 5.5 Hz, 2H), 3.47 (s, 3H), 3.38 (s, 3H), 1.45 (s, 12H). 13 C NMR (100 MHz, CDCl3) d 186.21, 164.51, 162.00, 160.90, 157.95, 154.94, 147.78, 135.76, 135.26, 135.14, 131.12, 130.18, 129.99, 127.94, 127.09, 124.42, 122.31, 120.85, 117.64, 116.48, 111.42, 84.59, 50.75, 43.39, 39.31, 39.03, 38.70, 24.98. HRMS (ESI positive) calc. for C33H32BN3O8SNaþ, [MþNa]þ 644.1895, found 644.1895. 2.3. Cytotoxicity assay
Please cite this article as: Y. Du et al., Dual-site fluorescent probe for multi-response detection of ClO and H2O2 and bio-imaging, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.12.059
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with PBS 3 times. 2.5. Imaging in live liver tissues Live liver tissues were obtained from the just killed 14 old days C57/BL6 mice and placed into pre-cooling PBS to wash off the blood. Next, these tissues were cut into slices. In each set of experiments, the liver tissue slices were incubated with Geisha-1 (10 mM) for 1.5 h at 37 C and then washed with PBS buffer 3 times. The slices were subsequently incubated with NaClO (200 mM), H2O2 (1 mM) and NaClO (200 mM) þ H2O2 (1 mM) for 30 min, 1 h and 1 h respectively. Tissue imaging was carried out after washing the slices with PBS 3 times. 3. Results and discussion 3.1. Design and synthesis of probe Geisha-1 The design of Geisha-1 is based on the following considerations: (a) the coumarin and 1,8-naphthalimide units should be modified to have suitable overlap between the fluorescence of donor unit and the absorption of acceptor unit and combined with a suitable length of non-conjugated linker to achieve the FRET process, (b) the two recognition sites on the probe should be of great sensitivity and selectivity toward corresponding analytes, and (c) the probe should exhibit three different responses for ClO, H2O2 and ClO þ H2O2. Based on all above, we chose 7-hydroxycoumarin and 4-hydroxy1,8-naphthalimide as the donor and acceptor fluorophores to construct a FRET platform, due to the heavy overlap between the fluorescence of 7-hydroxycoumarin derivative CD and the absorption of 4-hydroxy-1,8-naphthalimide derivative ND (Fig. S1). Since ethylenediamine has been widely used in the construction of FRET probes because of its suitable length [44e46], it was chosen as the linker. In addition, considering the chemical similarity between ClO and H2O2, it is critical to select two different detection mechanisms to achieve excellent selectivity. Therefore, N,Ndimethylthiocarbamate was selected as the recognition group targeted at ClO, because the recognition reaction is driven by Clþ that is available from HClO [47e49]. Borate was selected as the recognition group toward H2O2 due to its high reactivity and specificity for H2O2 [50e54]. As a result, a dual-site probe Geisha-1 was synthesized by the route shown in Scheme 1, and its structure was confirmed by 1H NMR, 13C NMR and HRMS (ESI) (Figs. S18e20). 3.2. Three responses of Geisha-1 to ClO, H2O2 and ClO þ H2O2 With the probe in hand, we firstly checked the responses of
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Geisha-1 toward ClO, H2O2 and ClO þ H2O2 respectively, and found that three different response channels came into being as expected (as shown in Scheme 2, Channel 1: lex ¼ 400 nm, lem ¼ 452 nm; Channel 2: lex ¼ 450 nm, lem ¼ 550 nm; Channel 3 based on FRET mechanism: lex ¼ 400 nm, lem ¼ 550 nm). As shown in Fig. 1a, alone addition of NaClO produces a response corresponding to Channel 1 (blue line, fluorescence quantum yield: F ¼ 0.0778). Similarly, when we just added H2O2, the response is observed to be consistent with the Channel 2 (Fig. 1b, purple line, fluorescence quantum yield: F ¼ 0.1992). When NaClO and H2O2 were simultaneously added, as shown in Fig. 1a, at 452 nm, the fluorescence emission decreases drastically (red line) compared with that of Channel 1, but it increases at 550 nm compared with the purple line. These changes suggest that the FRET process occurs, and the response corresponds to Channel 3 (Fig. 1a, red line, fluorescence quantum yield: F ¼ 0.1045). In addition, the signal of Channel 2 occurs owing to the presence of H2O2 (Fig. 1b, red line, fluorescence quantum yield: F ¼ 0.1390), so that the detection of NaClO and H2O2 do not interfere with each other because of the presence of both Channel 2 and Channel 3. Thus, Geisha-1 can simultaneously detect ClO and H2O2 via three different response mechanisms. 3.3. Sensitivity of probe Geisha-1 To explore the sensitivity of probe Geisha-1 to ClO or H2O2, UVevis titration of Geisha-1 toward ClO (0e300 mM) or H2O2 (0e1000 mM) were measured firstly. As shown in Fig. S2, in the absence of ClO and H2O2, Geisha-1 displays a strong absorption band at 344 nm. After adding NaClO (ClO donor), the absorption peak at 344 nm decreases, and two new absorption peaks at 373 and 404 nm appear and gradually increase due to the reversible equilibrium between 7-hydroxycoumarin unit and its conjugate base. When Geisha-1 is incubated with H2O2, the absorption peak at 344 nm decreases with a slight blue shift, while a new absorption band at 450 nm appears with an increase in absorbance. It is worth noting that the new absorption peak occurred by adding H2O2 does not appear when ClO is added, and vice versa, which indicate that there is no interference between the responses of ClO and H2O2. Then fluorescent spectral response of Geisha-1 to ClO or H2O2 was evaluated (Fig. 2). The probe displays almost no fluorescence emission under excitation at both 400 (fluorescence quantum yield: F ¼ 0.0282) and 450 nm (fluorescence quantum yield: F ¼ 0.0106), owing to the quenching effect of the two recognition groups. As shown in Fig. 2a, when adding NaClO, a turn-on fluorescent emission at 452 nm appears under excitation at 400 nm. Its fluorescence intensity increases drastically and reaches approximately 213-fold when added 300 mM NaClO. According to the inset
Scheme 1. Synthesis route of probe Geisha-1.
Please cite this article as: Y. Du et al., Dual-site fluorescent probe for multi-response detection of ClO and H2O2 and bio-imaging, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.12.059
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Scheme 2. Three responses of Geisha-1 to ClO, H2O2 and ClO þ H2O2.
Fig. 1. Fluorescence spectra of probe Geisha-1 (10 mM) in the absence or presence of NaClO (150 mM, 10 min), H2O2 (1000 mM, 70 min) and NaClO þ H2O2 (150 mM, 1000 mM, 70 min) (a). with the excitation at 400 nm and (b). with the excitation at 450 nm.
picture of Fig. 2a, a gradually enhanced bluish violet fluorescence can be observed under UV light at 365 nm. In addition, the fluorescence intensity at 452 nm exhibits a good linear relationship with the concentration of ClO within the range of 0e150 mM, and the detection limit for ClO is calculated to be 28.2 nM (3s/k) (inset of Fig. 2a), indicating that Geisha-1 is able to quantitatively detect ClO. As shown in Fig. 2b, similar to the detection of ClO, when excitated at 450 nm, the addition of H2O2 leads to a distinct turn-on fluorescence emission at 550 nm, and the fluorescence intensity reaches almost 260-fold as the concentration of H2O2 increasing from 0 to 1000 mM. The fluorescence color changes from colorless to yellow as shown in the inset picture of Fig. 2b. Besides, there is a good linearity between the fluorescence intensity at 550 nm and the concentration of H2O2 within the range of 0e90 mM, with the detection limit of 64.6 nM (3s/k) (inset of Fig. 2b), indicating that the probe could achieve quantitative detection of H2O2. These results suggest that Geisha-1 is able to distinguish ClO and H2O2
upon two fluorescence signals. 3.4. Effects of sensing time and pH Sensing time is an important factor to evaluate the performance of fluorescent probe. The time-dependent fluorescent response of Geisha-1 toward ClO and H2O2 are investigated first. With the excitation wavelength at 400 nm, the fluorescence intensity at 452 nm increases rapidly and reaches a plateau within 180 s after adding NaClO (Fig. S3a). Moreover, with the excitation wavelength at 450 nm, the fluorescence intensity at 550 nm gradually increases after adding H2O2 and reaches a plateau at 70 min (Fig. S3b). Furthermore, the dynamic change of the fluorescence upon the reaction of Geisha-1 with ClO and H2O2 is shown in Fig. S4. In the initial stage of the response process, with the excitation wavelength at 400 nm, a strong fluorescence emission occurs at 452 nm and a weak fluorescence emission occurs at 550 nm. As time goes by, the
Please cite this article as: Y. Du et al., Dual-site fluorescent probe for multi-response detection of ClO and H2O2 and bio-imaging, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.12.059
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Fig. 2. (a). Fluorescence spectra of probe Geisha-1 (10 mM) in the presence of NaClO (0e300 mM). Inset: Relationship between the fluorescence intensity at 452 nm and NaClO concentration. lex ¼ 400 nm. The inset picture shows the fluorescent changes in probe Geisha-1 upon addition of NaClO under UV light at 365 nm. Each datum was acquired 10 min after NaClO addition. (b). Fluorescence spectra of probe Geisha-1 (10 mM) in the presence of H2O2 (0e1000 mM). lex ¼ 450 nm. Inset: Relationship between the fluorescence intensity at 452 nm and H2O2 concentration. The inset picture shows the fluorescent changes in probe Geisha-1 upon addition of H2O2 under UV light at 365 nm. Each datum was acquired 70 min after H2O2 addition.
fluorescence intensity at 452 nm gradually weakens, while the fluorescence intensity at 550 nm gradually increases, which is attributed to the occurrence of FRET process. In order to determine the suitable pH range, the effects of pH were investigated afterwards. In the absence of analytes, Geisha-1 exhibits almost no fluorescence within the pH range 4.0e9.0 (Fig. S5, black lines).
When ClO or H2O2 is added, the corresponding fluorescence intensity displays a distinct enhancement within pH 6.0e9.0 for ClO (Fig. S5a, red line) and pH 7.0e9.0 for H2O2 (Fig. S5b, red line). These indicate that the probe is suitable to detect ClO and H2O2 under physiological conditions.
Please cite this article as: Y. Du et al., Dual-site fluorescent probe for multi-response detection of ClO and H2O2 and bio-imaging, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.12.059
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Fig. 3. (a). Fluorescence response of Geisha-1 (10 mM) to analytes (1000 mM). Analytes: 0. Blank, 1. Glu, 2. Cys, 3. Arg, 4. Gly, 5. Met, 6.Lys, 7. Hcy, 8. Trp, 9. Fe3þ, 10. Al3þ, 11. Fe2þ, 12. Cd 2þ, 13. Ba2þ, 14. Cu2þ, 15. Zn2þ, 16. Ca2þ, 17. Mg2þ, 18. Naþ, 19. Kþ, 20. NH4þ, 21. F, 22. Cl, 23. Br, 24. I, 25. SCN, 26. S2O32-, 27. S2O52-, 28. HSO4, 29.HSO3, 30. SO32-, 31. HS, 32. S2-, 33. TBHP, 34. H2O2, 35. ∙OH, 36. ∙OtBu, 37. NO, 38. ONOO, 39. 1O2, 40. NaClO; lex ¼ 400 nm and lem ¼ 452 nm. (b). Fluorescence response of Geisha-1 (10 mM) to analytes (1000 mM). Analytes:0. Blank, 1. Glu, 2. Cys, 3. Arg, 4. Gly, 5. Met, 6.Lys, 7. Hcy, 8. Trp, 9. Fe3þ, 10. Al3þ, 11. Fe2þ, 12. Cd 2þ, 13. Ba2þ, 14. Cu2þ, 15. Zn2þ, 16. Ca2þ, 17. Mg2þ, 18. Naþ, 19. Kþ, 2t 1 22 220. NHþ 4 , 21. F , 22. Cl , 23. Br , 24. I , 25. SCN , 26. S2O3 , 27. S2O5 , 28. HSO4 , 29.HSO3 , 30. SO3 , 31. HS , 32. S , 33. TBHP, 34. ∙OH, 35. NaClO, 36. ∙O Bu, 37. NO, 38. ONOO , 39. O2, 40. H2O2; lex ¼ 450 nm and lem ¼ 550 nm.
Fig. 4. Confocal fluorescence imaging of Hela Cells treated with probe Geisha-1 (10 mM) in the absence (aed) or presence of NaClO (eeh), H2O2 (iel) and NaClO þ H2O2 (mep). Channel 1 (lex ¼ 405 nm, lem ¼ 450 nm), Channel 2 (lex ¼ 488 nm, lem ¼ 550 nm) and Channel 3 (lex ¼ 405 nm, lem ¼ 550 nm). Scale bar: 10 mm.
Please cite this article as: Y. Du et al., Dual-site fluorescent probe for multi-response detection of ClO and H2O2 and bio-imaging, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.12.059
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3.5. Selectivity of the probe Geisha-1 High selectivity is necessary for dual-site probe to distinguish the two analytes. So we studied the fluorescence responses of Geisha-1 to various analytes including amino acids (Glu, Arg, Glv, Met, Lys, Trp), cations (Fe3þ, Al3þ, Fe2þ, Cd2þ, Ba2þ, Cu2þ, Zn2þ, Ca2þ, 22 Mg2þ, Naþ, Kþ, NHþ 4 ), anions (F , Cl , Br , I , SCN , S2O3 , S2O5 , 2 2HSO4 ), reactive sulfur species (Cys, Hcy, HSO3 , SO3 , HS , S ) and reactive oxygen/nitrogen species (NaClO, H2O2, TBHP, ∙OH, ∙OtBu, ONOO, 1O2, NO) in the excitation conditions of 400 and 450 nm respectively. Fig. 3a indicates that only NaClO can significantly enhance the fluorescence intensity at 452 nm (lex ¼ 400 nm) while other analytes don’t. Similarly, as shown in Fig. 3b, although some ROS can slightly enhance the fluorescence intensity at 550 nm, their effects are not comparable with that of H2O2. These results indicate that the two recognition sites of Geisha-1 exhibit great selectivity for ClO and H2O2 respectively, which guarantees the simultaneously and highly selective detection of ClO and H2O2. 3.6. The study on response mechanism of Geisha-1 toward ClO þ H2O2 The proposed response mechanism of Geisha-1 to ClO and H2O2 was shown in Scheme 2. In order to further prove the mechanism, the major product generated from the reaction of Geisha-1, ClO and H2O2 was purified by silica gel chromatography (ethyl acetate/methanol ¼ 1:1, v/v) and determined by 1H NMR analysis. No signal peaks corresponding to the protons of N,N-
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dimethylthiocarbamate and borate were found in the high-field region of 1H NMR spectrum (Fig. S21), suggesting the removal of the recognition groups. Furthermore, the HRMS analysis of the product was also performed (Fig. S22), where the peak at m/ z ¼ 467.0850 corresponding to the proposed product ([M þ Na]þ: 467.0850) was clearly observed. Thus, the results are in line with the proposed mechanism. 3.7. Fluorescence imaging of ClO and H2O2 in living cells Encouraged by the above promising results, we further applied the probe to image ClO and H2O2 in living cells. The cytotoxicity test was performed first and indicated that Geisha-1 has low toxicity under the experimental conditions at a concentration of 10 mM (Fig. S6). Hela cells were selected as the bioassay model to study the performance of Geisha-1. As expected, the Hela cells incubated only with Geisha-1 presented almost no fluorescence signal in all the three channels (Fig. 4bed). The fluorescence (blue) was observed only at Channel 1 when the living cells stained with Geisha-1 were incubated with NaClO for 30 min (Fig. 4feh), and the fluorescence (red) was obtained only at Channel 2 when incubated with H2O2 for 1 h (Fig. 4i-l). If the cells were incubated together with NaClO and H2O2 for 1 h, the fluorescence based on FRET mechanism emerged at Channel 3 (Fig. 4o) and the red fluorescence image was obtained in the Channel 2 (Fig. 4p). Interestingly, the fluorescence from Channel 3 is mainly distributed in the cytoplasm (Fig. 4o) and the fluorescence from Channel 2 is distributed in the entire cells (Fig. 4p). The difference indicates that ClO is mainly
Fig. 5. Confocal fluorescence imaging of mouse liver tissues stained with probe Geisha-1 (10 mM) in the absence (aee) or presence of NaClO (fej), H2O2 (keo) and NaClO þ H2O2 (pet). Channel 1 (lex ¼ 405 nm, lem ¼ 450 nm), Channel 2 (lex ¼ 488 nm, lem ¼ 550 nm) and Channel 3 (lex ¼ 405 nm, lem ¼ 550 nm). Scale bar: 25 mm.
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distributed in the cytoplasm and H2O2 is distributed throughout the cells, which can also be supported by Fig. 4f and l. Above results suggested that the probe is cell membrane permeable and can be used to detect ClO and H2O2 in living cells.
3.8. Fluorescence imaging of ClO and H2O2 in mouse liver tissues In order to investigate the application of probe Geisha-1 to image ClO and H2O2 in live tissues, we carried out fluorescence imaging experiments in mouse liver tissues. Similar to the results of cell imaging, the tissues incubated only with Geisha-1 presented almost no fluorescence signal in all the three channels (Fig. 5aee). When the tissues stained with Geisha-1 were incubated with NaClO, H2O2 and NaClO þ H2O2, the fluorescence emerged at Channel 1 (Fig. 5g), Channel 2 (Fig. 5n), and Channel 2 þ Channel 3 (Fig. 5r and s), respectively. These results verified that probe Geisha-1 exhibits excellent tissue penetration and detects ClO and H2O2 promisingly in live tissues.
[7]
[8] [9] [10]
[11]
[12] [13]
[14]
[15]
4. Conclusion In summary, we developed the first FRET-based dual-site fluorescent probe Geisha-1 for the detection of ClO, H2O2 and ClO þ H2O2 with three different responses. Geisha-1 exhibits excellent sensitivity (28.2 nM for ClO and 64.6 nM for H2O2) and high selectivity toward both ClO and H2O2. In addition, it was successfully applied to detect the intracellular and intra-tissue ClO and H2O2 by fluorescence turn-on mode, and the imaging experiments verify its relatively low cytotoxicity and satisfactory cellmembrane and tissue permeability, as well as its prospect in disease diagnosis or study of related physiological and pathological processes. Moreover, our strategy for the design of dual-site probe with three responses provides a valuable and flexible approach to construct fluorescent probes for simultaneous detection of two important biomolecules.
[16]
[17]
[18] [19]
[20]
[21]
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Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. This work was supported by the National Natural Science Foundation of China (Grant 21576194 and 21808161). We are grateful to professor Haixia Chen (Tianjin University) for their helpful suggestions and assistance with the cell imaging experiments.
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[26] [27]
Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.aca.2019.12.059. Acknowledgement.
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Dual-site fluorescent probe for multi-response detection of ClO and H2O2 and bio-imaging Yuchao Du a, Bowei Wang a, b, c, **, Di Jin a, b, Mingrui Li a, Yang Li a, b, c, Xilong Yan a, b, c, Xueqin Zhou a, b, c, Ligong Chen a, b, c, * a b c
School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300350, PR China Collaborative Innovation Center of Chemical Science and Engineering, Tianjin, PR China Tianjin Engineering Research Center of Functional Fine Chemicals, Tianjin, PR China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
The first dual-site fluorescent probe (Geisha-1) for the simultaneous detection of ClO and H2O2 was developed. The probe presented three different fluorescence response modes toward ClO, H2O2 and ClO þ H2O2 respectively. The probe could achieve quantitative detection of ClO and H2O2 with both excellent sensitivity and high selectivity. Geisha-1 was successfully applied to detect intracellular and intra-tissue ClO and H2O2.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 September 2019 Received in revised form 2 December 2019 Accepted 19 December 2019 Available online xxx
Hypochlorite (ClO) and hydrogen peroxide (H2O2) commonly coexist in organism and are involved in the same physiological and pathological processes. So it is of great importance to develop fluorescent probes to detect both simultaneously. Herein, we reported the first dual-site fluorescent probe (Geisha1) for the quantitative detection of ClO and H2O2. This probe is constructed by chemically grafting N,Ndimethylthiocarbamate and borate to a fluorescence resonance energy transfer (FRET) platform. As a result, Geisha-1 not only presents three different responses to ClO, H2O2, and ClO þ H2O2 (the coexistence of ClO and H2O2) with high sensitivity and selectivity, but also exhibits low toxicity and cell membrane and tissue permeability, and it was further successfully applied to image ClO and H2O2 in living cells and tissues. Thus, Geisha-1 provides a promising application prospect in biological systems and an alternative strategy for the construction of dual-site fluorescent probes aiming at the multiresponse detection of other biologically relevant analytes. © 2019 Elsevier B.V. All rights reserved.
Keywords: Fluorescent probe Dual-site Hypochlorite Hydrogen peroxide Bio-imaging
* Corresponding author. School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300350, PR China. ** Corresponding author. School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300350, PR China. E-mail addresses: [email protected] (B. Wang), [email protected] (L. Chen).
1. Introduction Reactive oxygen species (ROS) is a class of free radicals or nonradical oxygenated molecules those are highly reactive to biomolecules [1]. ROS are normally generated along with oxygen
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spectrometer with tetramethylsilane (TMS) as an internal standard. High-resolution mass spectra were recorded with a Bruker Daltonics microTOF-Q II instrument (ESI). All pH measurements were carried out with a Sartorius basic pH-meter PB-10. UVevis absorption spectra were obtained by using an L8 spectrophotometer. Fluorescence spectra were measured on a Hitachi F-2500 fluorescence spectrophotometer. Fluorescence quantum yields were determined by a FluoroMax-4 (Horiba Jobin Yvon) fluorometer equipped with an integrated sphere. For the fluorescence measurements, the excitation and emission slit widths were both 10 nm and the excitation wavelengths were 400/450 nm. Standard cell viability protocols (MTT assay) was taken on a spectra Max 190 microplate reader. The cell imaging was performed on a Leica TCS SP8 confocal laser scanning microscopy.
metabolism in the human immune system and plays an important role in cell signaling, migration and anti-inflammatory regulation [2,3]. As two important members of the ROS family, both hypochlorite (ClO) and hydrogen peroxide (H2O2) play a key regulatory role in normal cells [4e9]. However, excessive or misplaced ClO and H2O2 will cause oxidative stress and related diseases [10e14]. Therefore, monitoring of intracellular ClO and H2O2 is of great significance. Furthermore, it is meaningful to simultaneously detect ClO and H2O2, because they coexist in some cells and both synergistically regulate certain physiological processes or are involved in the same pathological processes. For example, in the immune system, H2O2 serves as a signal transducer, which activates macrophages to release ClO that can directly kill microorganisms [15e17]. In addition, increasing evidences have shown that both ClO and H2O2 are involved in neurodegenerative Alzheimer’s and Parkinson’s diseases, cardiovascular diseases and certain cancers [18e22]. However, distinguishing ClO and H2O2 is still challenging due to their similar chemical properties. Therefore, it is urgent to develop a method to simultaneously detect ClO and H2O2 with good selectivity, for the better understanding their complicated interrelationship and related physiological and pathological processes. Although colorimetric [23,24], electrochemical [25,26], and chromatographic [27,28] analytical methods, have been adopted to detect ClO or H2O2, the fluorescence analysis stood out to be the most eye-catching one due to its low cost, high sensitivity, noninvasive, and deep imaging ability [29e32]. To date, a number of probes toward ClO or H2O2 have been reported [33e37]. However, none of them can detect ClO and H2O2 synchronously. Simultaneous using of two corresponding probes seems to be a viable strategy, but it is inevitably subject to the cross-talk, stronger invasive effect, and different location and metabolism, making imaging more complicated and difficult to control. The dual-site probes proposed recently [38e43], which were equipped with two flexible recognition sites to overcome the above problems, are especially suitable for the simultaneous detection of two biomolecules. However, to the best of our knowledge, the dual-site probe capable of simultaneously imaging ClO and H2O2 has not been reported. Furthermore, the reported dual-site probes always just showed two responses toward different analytes respectively and lack of a third response to the coexisting analytes, which may cause the interference and distortion of the signal. Therefore, it is necessary to introduce a third response to the dual-site probe, namely two sites with triple responses, which is capable of differentiating ClO, H2O2, and ClO þ H2O2 (the coexistence of ClO and H2O2). Fluorescence resonance energy transfer (FRET) allows the probe to emit the fluorescence of acceptor fluorophore at the excitation wavelength of donor fluorophore, which makes it an effective tool to introduce a third response. In short, a coumarin-naphthalimide FRET platform was constructed and a dual-site fluorescent probe (Geisha-1) with three different responses to ClO, H2O2, and ClO þ H2O2 was developed.
Hela cells were used for cytotoxicity assay of probe Geisha-1 based on the standard cell viability protocols (MTT assay). Hela cells were grown in Dulbecco’s modified eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) in an atmosphere of 5% CO2 and 95% air at 37 C. Before the experiments, the cells were plated into a 96-well plate at a density of 5 105 cells per well and allowed to adhere for 24 h. Subsequently, the medium was replaced with fresh medium including 5, 10, 15, and 20 mM of Geisha-1. The cells that has not been treated with Geisha-1 were used as the blank control. After further incubation for 24 h, MTT solution (5 mg/ mL in PBS, 10 mM) was added to each well and incubation for another 4 h. Then the MTT solution was removed and 100 mL DMSO was added to dissolve the formazan crystals. After shaking for 10 min, the absorbance was measured at 570 nm using a microplate reader (spectra Max 190).
2. Experimental
2.4. Cell imaging
2.1. Materials and general methods
The Hela Cells were grown in DEME supplemented with 12% FBS in an atmosphere of 5% CO2 and 95% air at 37 C. The cells were plated on 35 mm glass bottom dishes and allowed to grow for 24 h. Before the experiments, cells were washed with PBS buffer. In each set of experiments, cells were incubated with Geisha-1 (10 mM) for 1.5 h at 37 C and then washed with PBS buffer 3 times. The cells were subsequently incubated with NaClO (200 mM), H2O2 (1 mM) and NaClO (200 mM) þ H2O2 (1 mM) for 30 min, 1 h and 1 h respectively. Cell imaging was carried out after washing the cells
All reagents and solvents were purchased from commercial sources and used without further purification unless otherwise noted. Deionized water was used throughout this work. Reactions were monitored by thin-layer chromatography (TLC) on silica gelprecoated glass plates. Silica gel (particle size 200e400 mesh) was used for column chromatography. 1H NMR and 13C NMR spectra were recorded with a Bruker Avance III 400 MHz
2.2. Synthesis of the probe Geisha-1 Compound 4 (1.64 g, 3.0 mmol) and triethylamine (0.90 g, 9.0 mmol) were dissolved in 30 mL anhydrous dichloromethane, and then a solution of N,N-dimethylaminothioformyl chloride (2.20 g, 18.0 mmol) in 3 mL anhydrous dichloromethane was added dropwise at 0 C. After stirring below 0 C for 90 min, the mixture was stirred at room temperature for 10 h. The solvent was removed by evaporation and the residue was purified by silica gel column chromatography (dichloromethane/methanol ¼ 100:1, v/v) to afford the desired Geisha-1 as a white solid (1.17 g, 60.8% yield). 1H NMR (400 MHz, CDCl3), d 9.12 (d, J ¼ 8.3 Hz, 1H), 8.96 (s, 1H), 8.83 (s, 1H), 8.60 (dd, J ¼ 14.0, 7.2 Hz, 2H), 8.29 (d, J ¼ 7.2 Hz, 1H), 7.77 (t, J ¼ 7.8 Hz, 1H), 7.65 (d, J ¼ 8.9 Hz, 1H), 7.11 (s, 2H), 4.53 (t, J ¼ 5.4 Hz, 2H), 3.88 (d, J ¼ 5.5 Hz, 2H), 3.47 (s, 3H), 3.38 (s, 3H), 1.45 (s, 12H). 13 C NMR (100 MHz, CDCl3) d 186.21, 164.51, 162.00, 160.90, 157.95, 154.94, 147.78, 135.76, 135.26, 135.14, 131.12, 130.18, 129.99, 127.94, 127.09, 124.42, 122.31, 120.85, 117.64, 116.48, 111.42, 84.59, 50.75, 43.39, 39.31, 39.03, 38.70, 24.98. HRMS (ESI positive) calc. for C33H32BN3O8SNaþ, [MþNa]þ 644.1895, found 644.1895. 2.3. Cytotoxicity assay
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with PBS 3 times. 2.5. Imaging in live liver tissues Live liver tissues were obtained from the just killed 14 old days C57/BL6 mice and placed into pre-cooling PBS to wash off the blood. Next, these tissues were cut into slices. In each set of experiments, the liver tissue slices were incubated with Geisha-1 (10 mM) for 1.5 h at 37 C and then washed with PBS buffer 3 times. The slices were subsequently incubated with NaClO (200 mM), H2O2 (1 mM) and NaClO (200 mM) þ H2O2 (1 mM) for 30 min, 1 h and 1 h respectively. Tissue imaging was carried out after washing the slices with PBS 3 times. 3. Results and discussion 3.1. Design and synthesis of probe Geisha-1 The design of Geisha-1 is based on the following considerations: (a) the coumarin and 1,8-naphthalimide units should be modified to have suitable overlap between the fluorescence of donor unit and the absorption of acceptor unit and combined with a suitable length of non-conjugated linker to achieve the FRET process, (b) the two recognition sites on the probe should be of great sensitivity and selectivity toward corresponding analytes, and (c) the probe should exhibit three different responses for ClO, H2O2 and ClO þ H2O2. Based on all above, we chose 7-hydroxycoumarin and 4-hydroxy1,8-naphthalimide as the donor and acceptor fluorophores to construct a FRET platform, due to the heavy overlap between the fluorescence of 7-hydroxycoumarin derivative CD and the absorption of 4-hydroxy-1,8-naphthalimide derivative ND (Fig. S1). Since ethylenediamine has been widely used in the construction of FRET probes because of its suitable length [44e46], it was chosen as the linker. In addition, considering the chemical similarity between ClO and H2O2, it is critical to select two different detection mechanisms to achieve excellent selectivity. Therefore, N,Ndimethylthiocarbamate was selected as the recognition group targeted at ClO, because the recognition reaction is driven by Clþ that is available from HClO [47e49]. Borate was selected as the recognition group toward H2O2 due to its high reactivity and specificity for H2O2 [50e54]. As a result, a dual-site probe Geisha-1 was synthesized by the route shown in Scheme 1, and its structure was confirmed by 1H NMR, 13C NMR and HRMS (ESI) (Figs. S18e20). 3.2. Three responses of Geisha-1 to ClO, H2O2 and ClO þ H2O2 With the probe in hand, we firstly checked the responses of
3
Geisha-1 toward ClO, H2O2 and ClO þ H2O2 respectively, and found that three different response channels came into being as expected (as shown in Scheme 2, Channel 1: lex ¼ 400 nm, lem ¼ 452 nm; Channel 2: lex ¼ 450 nm, lem ¼ 550 nm; Channel 3 based on FRET mechanism: lex ¼ 400 nm, lem ¼ 550 nm). As shown in Fig. 1a, alone addition of NaClO produces a response corresponding to Channel 1 (blue line, fluorescence quantum yield: F ¼ 0.0778). Similarly, when we just added H2O2, the response is observed to be consistent with the Channel 2 (Fig. 1b, purple line, fluorescence quantum yield: F ¼ 0.1992). When NaClO and H2O2 were simultaneously added, as shown in Fig. 1a, at 452 nm, the fluorescence emission decreases drastically (red line) compared with that of Channel 1, but it increases at 550 nm compared with the purple line. These changes suggest that the FRET process occurs, and the response corresponds to Channel 3 (Fig. 1a, red line, fluorescence quantum yield: F ¼ 0.1045). In addition, the signal of Channel 2 occurs owing to the presence of H2O2 (Fig. 1b, red line, fluorescence quantum yield: F ¼ 0.1390), so that the detection of NaClO and H2O2 do not interfere with each other because of the presence of both Channel 2 and Channel 3. Thus, Geisha-1 can simultaneously detect ClO and H2O2 via three different response mechanisms. 3.3. Sensitivity of probe Geisha-1 To explore the sensitivity of probe Geisha-1 to ClO or H2O2, UVevis titration of Geisha-1 toward ClO (0e300 mM) or H2O2 (0e1000 mM) were measured firstly. As shown in Fig. S2, in the absence of ClO and H2O2, Geisha-1 displays a strong absorption band at 344 nm. After adding NaClO (ClO donor), the absorption peak at 344 nm decreases, and two new absorption peaks at 373 and 404 nm appear and gradually increase due to the reversible equilibrium between 7-hydroxycoumarin unit and its conjugate base. When Geisha-1 is incubated with H2O2, the absorption peak at 344 nm decreases with a slight blue shift, while a new absorption band at 450 nm appears with an increase in absorbance. It is worth noting that the new absorption peak occurred by adding H2O2 does not appear when ClO is added, and vice versa, which indicate that there is no interference between the responses of ClO and H2O2. Then fluorescent spectral response of Geisha-1 to ClO or H2O2 was evaluated (Fig. 2). The probe displays almost no fluorescence emission under excitation at both 400 (fluorescence quantum yield: F ¼ 0.0282) and 450 nm (fluorescence quantum yield: F ¼ 0.0106), owing to the quenching effect of the two recognition groups. As shown in Fig. 2a, when adding NaClO, a turn-on fluorescent emission at 452 nm appears under excitation at 400 nm. Its fluorescence intensity increases drastically and reaches approximately 213-fold when added 300 mM NaClO. According to the inset
Scheme 1. Synthesis route of probe Geisha-1.
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Scheme 2. Three responses of Geisha-1 to ClO, H2O2 and ClO þ H2O2.
Fig. 1. Fluorescence spectra of probe Geisha-1 (10 mM) in the absence or presence of NaClO (150 mM, 10 min), H2O2 (1000 mM, 70 min) and NaClO þ H2O2 (150 mM, 1000 mM, 70 min) (a). with the excitation at 400 nm and (b). with the excitation at 450 nm.
picture of Fig. 2a, a gradually enhanced bluish violet fluorescence can be observed under UV light at 365 nm. In addition, the fluorescence intensity at 452 nm exhibits a good linear relationship with the concentration of ClO within the range of 0e150 mM, and the detection limit for ClO is calculated to be 28.2 nM (3s/k) (inset of Fig. 2a), indicating that Geisha-1 is able to quantitatively detect ClO. As shown in Fig. 2b, similar to the detection of ClO, when excitated at 450 nm, the addition of H2O2 leads to a distinct turn-on fluorescence emission at 550 nm, and the fluorescence intensity reaches almost 260-fold as the concentration of H2O2 increasing from 0 to 1000 mM. The fluorescence color changes from colorless to yellow as shown in the inset picture of Fig. 2b. Besides, there is a good linearity between the fluorescence intensity at 550 nm and the concentration of H2O2 within the range of 0e90 mM, with the detection limit of 64.6 nM (3s/k) (inset of Fig. 2b), indicating that the probe could achieve quantitative detection of H2O2. These results suggest that Geisha-1 is able to distinguish ClO and H2O2
upon two fluorescence signals. 3.4. Effects of sensing time and pH Sensing time is an important factor to evaluate the performance of fluorescent probe. The time-dependent fluorescent response of Geisha-1 toward ClO and H2O2 are investigated first. With the excitation wavelength at 400 nm, the fluorescence intensity at 452 nm increases rapidly and reaches a plateau within 180 s after adding NaClO (Fig. S3a). Moreover, with the excitation wavelength at 450 nm, the fluorescence intensity at 550 nm gradually increases after adding H2O2 and reaches a plateau at 70 min (Fig. S3b). Furthermore, the dynamic change of the fluorescence upon the reaction of Geisha-1 with ClO and H2O2 is shown in Fig. S4. In the initial stage of the response process, with the excitation wavelength at 400 nm, a strong fluorescence emission occurs at 452 nm and a weak fluorescence emission occurs at 550 nm. As time goes by, the
Please cite this article as: Y. Du et al., Dual-site fluorescent probe for multi-response detection of ClO and H2O2 and bio-imaging, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.12.059
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Fig. 2. (a). Fluorescence spectra of probe Geisha-1 (10 mM) in the presence of NaClO (0e300 mM). Inset: Relationship between the fluorescence intensity at 452 nm and NaClO concentration. lex ¼ 400 nm. The inset picture shows the fluorescent changes in probe Geisha-1 upon addition of NaClO under UV light at 365 nm. Each datum was acquired 10 min after NaClO addition. (b). Fluorescence spectra of probe Geisha-1 (10 mM) in the presence of H2O2 (0e1000 mM). lex ¼ 450 nm. Inset: Relationship between the fluorescence intensity at 452 nm and H2O2 concentration. The inset picture shows the fluorescent changes in probe Geisha-1 upon addition of H2O2 under UV light at 365 nm. Each datum was acquired 70 min after H2O2 addition.
fluorescence intensity at 452 nm gradually weakens, while the fluorescence intensity at 550 nm gradually increases, which is attributed to the occurrence of FRET process. In order to determine the suitable pH range, the effects of pH were investigated afterwards. In the absence of analytes, Geisha-1 exhibits almost no fluorescence within the pH range 4.0e9.0 (Fig. S5, black lines).
When ClO or H2O2 is added, the corresponding fluorescence intensity displays a distinct enhancement within pH 6.0e9.0 for ClO (Fig. S5a, red line) and pH 7.0e9.0 for H2O2 (Fig. S5b, red line). These indicate that the probe is suitable to detect ClO and H2O2 under physiological conditions.
Please cite this article as: Y. Du et al., Dual-site fluorescent probe for multi-response detection of ClO and H2O2 and bio-imaging, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.12.059
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Fig. 3. (a). Fluorescence response of Geisha-1 (10 mM) to analytes (1000 mM). Analytes: 0. Blank, 1. Glu, 2. Cys, 3. Arg, 4. Gly, 5. Met, 6.Lys, 7. Hcy, 8. Trp, 9. Fe3þ, 10. Al3þ, 11. Fe2þ, 12. Cd 2þ, 13. Ba2þ, 14. Cu2þ, 15. Zn2þ, 16. Ca2þ, 17. Mg2þ, 18. Naþ, 19. Kþ, 20. NH4þ, 21. F, 22. Cl, 23. Br, 24. I, 25. SCN, 26. S2O32-, 27. S2O52-, 28. HSO4, 29.HSO3, 30. SO32-, 31. HS, 32. S2-, 33. TBHP, 34. H2O2, 35. ∙OH, 36. ∙OtBu, 37. NO, 38. ONOO, 39. 1O2, 40. NaClO; lex ¼ 400 nm and lem ¼ 452 nm. (b). Fluorescence response of Geisha-1 (10 mM) to analytes (1000 mM). Analytes:0. Blank, 1. Glu, 2. Cys, 3. Arg, 4. Gly, 5. Met, 6.Lys, 7. Hcy, 8. Trp, 9. Fe3þ, 10. Al3þ, 11. Fe2þ, 12. Cd 2þ, 13. Ba2þ, 14. Cu2þ, 15. Zn2þ, 16. Ca2þ, 17. Mg2þ, 18. Naþ, 19. Kþ, 2t 1 22 220. NHþ 4 , 21. F , 22. Cl , 23. Br , 24. I , 25. SCN , 26. S2O3 , 27. S2O5 , 28. HSO4 , 29.HSO3 , 30. SO3 , 31. HS , 32. S , 33. TBHP, 34. ∙OH, 35. NaClO, 36. ∙O Bu, 37. NO, 38. ONOO , 39. O2, 40. H2O2; lex ¼ 450 nm and lem ¼ 550 nm.
Fig. 4. Confocal fluorescence imaging of Hela Cells treated with probe Geisha-1 (10 mM) in the absence (aed) or presence of NaClO (eeh), H2O2 (iel) and NaClO þ H2O2 (mep). Channel 1 (lex ¼ 405 nm, lem ¼ 450 nm), Channel 2 (lex ¼ 488 nm, lem ¼ 550 nm) and Channel 3 (lex ¼ 405 nm, lem ¼ 550 nm). Scale bar: 10 mm.
Please cite this article as: Y. Du et al., Dual-site fluorescent probe for multi-response detection of ClO and H2O2 and bio-imaging, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.12.059
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3.5. Selectivity of the probe Geisha-1 High selectivity is necessary for dual-site probe to distinguish the two analytes. So we studied the fluorescence responses of Geisha-1 to various analytes including amino acids (Glu, Arg, Glv, Met, Lys, Trp), cations (Fe3þ, Al3þ, Fe2þ, Cd2þ, Ba2þ, Cu2þ, Zn2þ, Ca2þ, 22 Mg2þ, Naþ, Kþ, NHþ 4 ), anions (F , Cl , Br , I , SCN , S2O3 , S2O5 , 2 2HSO4 ), reactive sulfur species (Cys, Hcy, HSO3 , SO3 , HS , S ) and reactive oxygen/nitrogen species (NaClO, H2O2, TBHP, ∙OH, ∙OtBu, ONOO, 1O2, NO) in the excitation conditions of 400 and 450 nm respectively. Fig. 3a indicates that only NaClO can significantly enhance the fluorescence intensity at 452 nm (lex ¼ 400 nm) while other analytes don’t. Similarly, as shown in Fig. 3b, although some ROS can slightly enhance the fluorescence intensity at 550 nm, their effects are not comparable with that of H2O2. These results indicate that the two recognition sites of Geisha-1 exhibit great selectivity for ClO and H2O2 respectively, which guarantees the simultaneously and highly selective detection of ClO and H2O2. 3.6. The study on response mechanism of Geisha-1 toward ClO þ H2O2 The proposed response mechanism of Geisha-1 to ClO and H2O2 was shown in Scheme 2. In order to further prove the mechanism, the major product generated from the reaction of Geisha-1, ClO and H2O2 was purified by silica gel chromatography (ethyl acetate/methanol ¼ 1:1, v/v) and determined by 1H NMR analysis. No signal peaks corresponding to the protons of N,N-
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dimethylthiocarbamate and borate were found in the high-field region of 1H NMR spectrum (Fig. S21), suggesting the removal of the recognition groups. Furthermore, the HRMS analysis of the product was also performed (Fig. S22), where the peak at m/ z ¼ 467.0850 corresponding to the proposed product ([M þ Na]þ: 467.0850) was clearly observed. Thus, the results are in line with the proposed mechanism. 3.7. Fluorescence imaging of ClO and H2O2 in living cells Encouraged by the above promising results, we further applied the probe to image ClO and H2O2 in living cells. The cytotoxicity test was performed first and indicated that Geisha-1 has low toxicity under the experimental conditions at a concentration of 10 mM (Fig. S6). Hela cells were selected as the bioassay model to study the performance of Geisha-1. As expected, the Hela cells incubated only with Geisha-1 presented almost no fluorescence signal in all the three channels (Fig. 4bed). The fluorescence (blue) was observed only at Channel 1 when the living cells stained with Geisha-1 were incubated with NaClO for 30 min (Fig. 4feh), and the fluorescence (red) was obtained only at Channel 2 when incubated with H2O2 for 1 h (Fig. 4i-l). If the cells were incubated together with NaClO and H2O2 for 1 h, the fluorescence based on FRET mechanism emerged at Channel 3 (Fig. 4o) and the red fluorescence image was obtained in the Channel 2 (Fig. 4p). Interestingly, the fluorescence from Channel 3 is mainly distributed in the cytoplasm (Fig. 4o) and the fluorescence from Channel 2 is distributed in the entire cells (Fig. 4p). The difference indicates that ClO is mainly
Fig. 5. Confocal fluorescence imaging of mouse liver tissues stained with probe Geisha-1 (10 mM) in the absence (aee) or presence of NaClO (fej), H2O2 (keo) and NaClO þ H2O2 (pet). Channel 1 (lex ¼ 405 nm, lem ¼ 450 nm), Channel 2 (lex ¼ 488 nm, lem ¼ 550 nm) and Channel 3 (lex ¼ 405 nm, lem ¼ 550 nm). Scale bar: 25 mm.
Please cite this article as: Y. Du et al., Dual-site fluorescent probe for multi-response detection of ClO and H2O2 and bio-imaging, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.12.059
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distributed in the cytoplasm and H2O2 is distributed throughout the cells, which can also be supported by Fig. 4f and l. Above results suggested that the probe is cell membrane permeable and can be used to detect ClO and H2O2 in living cells.
3.8. Fluorescence imaging of ClO and H2O2 in mouse liver tissues In order to investigate the application of probe Geisha-1 to image ClO and H2O2 in live tissues, we carried out fluorescence imaging experiments in mouse liver tissues. Similar to the results of cell imaging, the tissues incubated only with Geisha-1 presented almost no fluorescence signal in all the three channels (Fig. 5aee). When the tissues stained with Geisha-1 were incubated with NaClO, H2O2 and NaClO þ H2O2, the fluorescence emerged at Channel 1 (Fig. 5g), Channel 2 (Fig. 5n), and Channel 2 þ Channel 3 (Fig. 5r and s), respectively. These results verified that probe Geisha-1 exhibits excellent tissue penetration and detects ClO and H2O2 promisingly in live tissues.
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4. Conclusion In summary, we developed the first FRET-based dual-site fluorescent probe Geisha-1 for the detection of ClO, H2O2 and ClO þ H2O2 with three different responses. Geisha-1 exhibits excellent sensitivity (28.2 nM for ClO and 64.6 nM for H2O2) and high selectivity toward both ClO and H2O2. In addition, it was successfully applied to detect the intracellular and intra-tissue ClO and H2O2 by fluorescence turn-on mode, and the imaging experiments verify its relatively low cytotoxicity and satisfactory cellmembrane and tissue permeability, as well as its prospect in disease diagnosis or study of related physiological and pathological processes. Moreover, our strategy for the design of dual-site probe with three responses provides a valuable and flexible approach to construct fluorescent probes for simultaneous detection of two important biomolecules.
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Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. This work was supported by the National Natural Science Foundation of China (Grant 21576194 and 21808161). We are grateful to professor Haixia Chen (Tianjin University) for their helpful suggestions and assistance with the cell imaging experiments.
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Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.aca.2019.12.059. Acknowledgement.
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Please cite this article as: Y. Du et al., Dual-site fluorescent probe for multi-response detection of ClO and H2O2 and bio-imaging, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.12.059