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Cite this: DOI: 10.1039/c4cc10382c Received 29th December 2014, Accepted 13th February 2015
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A two-photon fluorescent turn-on probe for nitroxyl (HNO) and its bioimaging application in living tissues† Kaibo Zheng,a Weiying Lin,*ab Dan Cheng,a Hua Chen,a Yong Liub and Keyin Liub
DOI: 10.1039/c4cc10382c www.rsc.org/chemcomm
The first two-photon fluorescent probe for specific detection of nitroxyl is designed and synthesized, and we have further demonstrated that the new two-photon fluorescent probe could be employed to image nitroxyl in living cells and tissues.
Nitroxyl (HNO), a one-electron-reduced form of NO, has recently received intense attention due to its biological functions and many potential pharmacological effects in mammalian systems.1 For example, HNO is used clinically as a deterrent for alcohol use, exhibits anti-cancer properties, shows promise for the treatment of heart failure, protects tissue against reperfusion injury, etc.1,2 Thus, it is of high significance to develop efficient methods for the detection of HNO in living systems. To date, several analytical techniques including colorimetric method,1a,3 EPR,4 HPLC,5 mass spectrometry6 and electrochemical analysis1a,7 have been developed for HNO detection. Although these methods provide sensitive analysis, they require complicated sample preparation, sophisticated instrumentation, or destruction of tissues or cells. Therefore, they are not suitable for applications in living systems. In recent years, fluorescent probes, as excellent molecular tools, have attracted increasing attention due to their high selectivity, high sensitivity, as well as real-time imaging, and they have been widely applied in the detection of biological molecules.8 A number of fluorescent probes for HNO based on the HNO-induced reduction of Cu(II) to Cu(I) have been developed.9 Alternatively, by taking advantage of the reaction of HNO with triarylphosphine to give the corresponding phosphine oxide and aza-ylide, fluorescent HNO probes have been engineered.10 However, the fluorescent probes for HNO reported are based on one-photon microscopy (OPM), which only show a shallow penetration depth. Moreover, the short excitation wavelengths a
State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, Hunan 410082, P. R. China b Institute of Fluorescent Probes for Biological Imaging, University of Jinan, Jinan, Shandong 250022, P. R. China. E-mail: [email protected] † Electronic supplementary information (ESI) available: Quantum yields, 1H NMR and 13C NMR data, MTT assay, and UV/vis absorption. See DOI: 10.1039/c4cc10382c
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may lead to photobleaching of probes and damage in cells and tissues, and thus limit their biological applications. By contrast, two-photon microscopy (TPM) is an attractive technique to study biomolecules in living cells and tissues. TPM, where fluorescence is triggered by two-photon excitation, is advantageous over conventional one-photon fluorescence microscopy.11 It can facilitate three-dimensional imaging of living tissues, reduce photodamage to biosamples, increase tissue penetration, and lower background fluorescence. These are favourable for imaging deep tissues. In spite of these advantages, to the best of our knowledge, no two-photon fluorescent HNO probes have been reported in the literature to date. Thus, it is of great interest to design two-photon fluorescent probes, which are suitable for monitoring HNO not only in living cells but also in living tissues. Recently, our group has reported a fluorescent dye GCTPOC, which contains a rigid oxygen-bridge and a hydroxyl group at the 2-position ( para-position). GCTPOC exhibits excellent two-photon properties with a two-photon cross-section (s) above 810 GM and a two-photon excitation action cross-section above 270 GM (excitation at 860 nm), indicating that the dye is potentially useful for bioimaging applications.12 GCTPOC has been demonstrated to be an efficient two-photon platform for designing two-photon probes with its tunable twophoton properties by modifications at the hydroxyl group. In this work, we developed GCTPOC-1 as the first two-photon fluorescent turn-on HNO probe using GCTPOC as the two-photon platform and triarylphosphine as the reactive site for HNO (Scheme 1). The two-photon probe GCTPOC-1 has a large fluorescence enhancement, which makes it attractive for imaging HNO in living tissues with deep tissue penetration. The starting compound GCTPOC was prepared according to a literature procedure.12 All new compounds were characterized by standard NMR spectroscopy and mass spectrometry. With GCTPOC-1 in hand, we examined its optical properties in the absence or presence of HNO (Angeli’s salt (AS) was used as the HNO source in all experiments). The free probe GCTPOC-1 (10 mM) exhibited a maximal absorption band at around 354 nm (e = 5051 M 1 cm 1) in pH 7.4, 25 mM PBS buffer (1% ethanol) at
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Scheme 1 Design and synthesis of the first two-photon fluorescent turn-on HNO probe GCTPOC-1.
ambient temperature (Fig. S1, ESI†). As anticipated, the free GCTPOC-1 showed relatively weak fluorescence (F = 0.001). However, upon treatment with HNO, a drastic enhancement in fluorescence intensity at around 512 nm was observed (Fig. 1). A maximal fluorescence enhancement (49-fold) was obtained when the concentration of AS reached 15 equiv. The detection limit for GCTPOC-1 was calculated to be 5.90 10 7 M (Fig. S2, ESI†), indicating that the probe is highly sensitive to HNO. To confirm the sensing process, we decided to study the product of GCTPOC-1 + HNO by both NMR spectroscopy and mass spectrometry. The product of GCTPOC-1 + HNO was isolated using a silica gel column and was then subjected to 1H NMR analysis. The 1H NMR of the resulting product is essentially identical to that of the authentic GCTPOC (The experiment section of the ESI†). In addition, the identity of the product of GCTPOC-1 + HNO was further characterized by mass spectrometry analysis (Fig. S3, ESI†). This is consistent with the previous report that HNO reacts with triarylphosphines to give the corresponding phosphine oxide.10 The time courses of the fluorescence intensities of GCTPOC-1 (5 mM) in the absence or presence of HNO (5, 10, and 15 equiv.) in pH 7.4, 25 mM PBS buffer (1% ethanol) are displayed in Fig. 2. Notably, a drastic enhancement in fluorescence intensities was observed within a few minutes, and a plateau was reached in 45 minutes in the presence of 5, 10 or 15 equiv. of AS. It suggests that the rapid fluorescence response of the probe may make it suitable for detection of HNO. We then examined the kinetic profiles of the reaction under pseudo-first-order conditions with a large excess of HNO (15 equiv.) over the probe GCTPOC-1
Fig. 1 Fluorescence spectra of GCTPOC-1 (5.0 mM) in pH 7.4 PBS buffer (1% ethanol) in the absence or presence of AS (0–100 mM). Inset: fluorescence intensity ratio (F/F0) changes at 512 nm of GCTPOC-1 (5.0 mM) with the amount of AS. The spectra were recorded after incubation of the probe with AS for 45 min.
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Fig. 2 Reaction-time profiles of GCTPOC-1 (5.0 mM) in the absence [’] or presence of AS (25 mM [ ], 50 mM [ ], and 75 mM [ ]). The fluorescence intensities at 512 nm were continuously monitored at time intervals in pH 7.4 PBS buffer (1% ethanol). Time points represent 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, and 60 min.
(5 mM) in pH 7.4, 25 mM PBS buffer (1% ethanol) at ambient temperature. The pseudo-first-order rate constant for HNO was determined to be k = 0.0487 min 1 (Fig. S4, ESI†). The effect of pH on the fluorescence response of GCTPOC-1 to HNO was investigated. As shown in Fig. S5 (ESI†), in the absence of HNO, only a very minor change in fluorescence intensity was observed in the free probe over a wide pH range of 5.0–8.5, indicating that the free probe was stable in the wide pH range. Upon treatment with AS, the maximal fluorescence signal was observed in the pH range of 7.0–8.5, indicating that the probe can be employed to detect HNO at a physiological pH of 7.4. However, the probe has relatively low sensitivity at lower pH. This may be improved in the future. The probe GCTPOC-1 was treated with various relevant analytes including anions, reactive oxygen species, reducing agents, small molecule thiols, and AS in pH 7.4, 25 mM PBS buffer (1% ethanol) to investigate the selectivity. As shown in Fig. 3, the addition of the representative species (ClO , H2O2, Fe3+, O2 , sodium ascorbate, N3 , NO2 , NO3 , Cys, Na2S (H2S), CORM-2 (CO), NO at 100 mM, and GSH at 1 mM) elicited essentially no fluorescence response for over 45 min. By contrast, importantly, introduction of AS (75 mM) caused a large fluorescence enhancement (49-fold) in 45 min. Notably, S-nitrosoglutathione (GSNO) at 100 mM triggered only a small fluorescence enhancement (o9 fold) and had nearly no interference for HNO detection. These data indicate that the probe is selective for HNO over other tested species. It is necessary to evaluate both the photostability and chemical stability of fluorescent probes designed to be used in biological environments. The photostability of GCTPOC-1 in pH 7.4, 25 mM PBS buffer (1% ethanol) at ambient temperature was measured by continuous irradiation with UV light (365 nm), and the results demonstrate that the emission profile of GCTPOC-1 has only minor changes after 1 h of irradiation (Fig. S6A, ESI†), indicating that this probe has sufficient photostability for potential biological use. In the experimental procedure, AS (the HNO source) was dissolved in 0.01 M NaOH solution in order to investigate
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Fig. 3 The fluorescence responses of the probe GCTPOC-1 (5.0 mM) to various relevant species (100 mM): 1. Blank; 2. ClO ; 3. H2O2; 4. sodium ascorbate; 5. O2 ; 6. NO; 7. NO2 ; 8. NO3 , 9. N3 ; 10. Fe3+; 11. CO; 12. H2S; 13. Cys; 14. GSH; 15. GSNO; and 16. AS, in pH 7.4, 25 mM PBS buffer (1% ethanol).
whether the NaOH solution (225 mL, 0.01 M NaOH) hydrolyzes the probe in pH 7.4, 25 mM PBS buffer (1% ethanol). There are many oxidizing and reducing reagents in biological settings. Thus, we further investigated the emission profiles of the probe GCTPOC-1 in the presence of the representative NaOH, oxidizing and reducing reagents. As a representative case, GCTPOC-1 in pH 7.4, 25 mM PBS buffer (1% ethanol) at ambient temperature was treated with H2O2, NaClO, Fe3+, Cys at 100 mM, NaOH at 0.75 mM or GSH at 1 mM for 1 h. As shown in Fig. S6B (ESI†), the emission profile of GCTPOC-1 is almost unchanged. Thus, these data suggest that the probe GCTPOC-1 displays sufficient chemical stability. In order to be useful as imaging agents, fluorescent probes should have low cytotoxicity. Thus, we investigated the potential toxicities of GCTPOC-1 against a representative cell line, HeLa cells. The living cells were incubated with various concentrations (5–50 mM) of GCTPOC-1 for 24 h, and then the cell viability was determined by the standard 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assays. The results indicate that GCTPOC-1 has no marked cytotoxicity at concentrations below 30 mM (Fig. S7, ESI†). Thus, the prominent features of GCTPOC-1 include good two-photon fluorescence properties, high sensitivity, excellent selectivity, relatively fast response, good functioning at physiological pH, and low cytotoxicity. These desirable attributes prompted us to further examine the suitability of the probe for visualizing HNO in living systems. Toward this end, the probe was incubated with HeLa cells in the absence or presence of AS, and the fluorescence images were recorded both in one-photon and two-photon modes. In the one-photon mode, the HeLa cells incubated with only the probe GCTPOC-1 (5.0 mM) for 20 min at 37 1C in PBS exhibited very weak fluorescence at the emission window of 470–570 nm (Fig. 4b). By sharp contrast, HeLa cells incubated with 5.0 mM GCTPOC-1 for 20 min, and then treated with AS (35 mM, 75 mM) displayed strong fluorescence (Fig. 4f and j). Consistently, in the two-photon mode (excitation at 780 nm), the HeLa cells incubated with only the probe showed almost no fluorescence (Fig. 4d). However, the cells pretreated with GCTPOC-1 and
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Fig. 4 One-photon fluorescent images: (a) bright-field image of living HeLa cells incubated with only GCTPOC-1 (5.0 mM) for 20 min; (b) Fluorescence image of (a); (e, i) bright-field image of living HeLa cells incubated with GCTPOC-1 (5.0 mM) for 20 min, then with AS (35 mM or 75 mM) for 45 min; (f, j) Fluorescence image of (e, i). Excitation at 405 nm. Two-photon fluorescence images: (c) bright-field image of living HeLa cells incubated with only GCTPOC-1 (5.0 mM) for 20 min; (d) fluorescence image of (c); (g, k) bright-field image of living HeLa cells incubated with GCTPOC-1 (5.0 mM) for 20 min, then with AS (35 mM or 75 mM) for 45 min; (h, l) fluorescence image of (g, k). Excitation at 780 nm. Scale bar = 20 mm.
further incubated with the AS (35 mM, 75 mM) exhibited bright fluorescence (Fig. 4h and l). The comparison among Fig. 4b, f, and j (Fig. 4d, h, and l) reveals a dose-dependent fluorescence enhancement. Thus, these data establish that the probe GCTPOC-1 is cell membrane permeable and capable of sensing HNO in living cells. When compared to the traditional one-photon fluorescence microscopy with emission in the visible region, the major advantage of two-photon fluorescence microscopy is that the latter is suitable for three-dimensional fluorescence imaging in thick living samples, for instance, living tissues, due to the localized excited volume. We were interested in exploiting the capability of the probe GCTPOC-1 in this aspect. Toward this end, living tissue slices (a thickness of 400 mm) of the liver were prepared and subjected to one-photon and two-photon fluorescence microscopic analyses. In a control experiment, the tissue slices incubated with only GCTPOC-1 (30 mM) for 40 min at 37 1C in PBS exhibited weak fluorescence in one-photon (Fig. S8, ESI†) and two-photon fluorescence modes (Fig. S9, ESI†) at the emission window of 520–570 nm. By contrast, when the tissue slices were incubated with 30 mM GCTPOC-1 for 40 min, and then treated with 1 mM AS, relatively significant fluorescence is only observed up to 70 mm in the one-photon fluorescence mode (Fig. S10, ESI†). However, in the two-photon mode (excitation at 780 nm), relatively significant fluorescence is observed up to 130 mm (Fig. 5). These data indicate that the probe GCTPOC-1 is capable of detecting HNO at 130 mm depth in living tissues using two-photon fluorescence microscopy, in accord with its good two-photon fluorescence properties. In summary, we have constructed the first two-photon fluorescent turn-on HNO probe GCTPOC-1 based on a two-photon platform with a large cross-section, GCTPOC, and a sensitive HNO
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Fig. 5 Two-photon fluorescence images of a fresh rat liver slice pretreated with 30 mM GCTPOC-1 and then with AS (1 mM) incubated at depths of approximately 0 B 150 mm with a magnification of 10. Excitation at 780 nm. Scale bar = 200 mm.
recognition site. The probe GCTPOC-1 exhibits desirable properties including high sensitivity, high selectivity, good functioning at physiological pH and low cytotoxicity. In particular, the probe shows a 49-fold enhancement in the presence of AS (75 mM). Importantly, we have demonstrated that the probe GCTPOC-1 is suitable for fluorescence imaging of HNO not only in living cells, but also in living tissues using two-photon fluorescence microscopy. Further applications of the two-photon probe for the investigation of the biological functions and pathological roles of HNO in living systems are under progress. This work was financially supported by the NSFC (21172063, 21472067) and the startup fund of University of Jinan.
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and D. A. Wink, Biochim. Biophys. Acta, 2009, 1787, 835–840; (b) J. M. Fukuto, C. J. Cisneros and L. Kinkade, J. Inorg. Biochem., 2013, 118, 201–208. (a) S. E. Bari, M. A. Martı´, V. T. Amorebieta, D. A. Estrin and F. Doctorovich, J. Am. Chem. Soc., 2003, 125, 15272–15273; (b) M. A. Martı´, S. E. Bari, D. A. Estrin and F. Doctorovich, J. Am. Chem. Soc., 2005, 127, 4680–4684. (a) D. G. Mitchell, R. W. Quine, M. Tseitlin, S. S. Eaton and G. R. Eaton, J. Magn. Reson., 2012, 214, 221–226; (b) U. Samuni, Y. Samuni and S. Goldstein, J. Am. Chem. Soc., 2010, 132, 8428–8432. (a) J. A. Reisz, C. N. Zink and S. B. King, J. Am. Chem. Soc., 2011, 133, 11675–11685; (b) S. Donzelli, M. G. Espey, D. D. Thomas, D. Mancardi, C. G. Tocchetti, L. A. Ridnour, N. Paolocci, S. B. King, K. M. Miranda, G. Lazzarino, J. M. Fukuto and D. A. Wink, Free Radical Biol. Med., 2006, 40, 1056–1066. M. R. Cline, C. Tu, D. N. Silverman and J. P. Toscano, Free Radical Biol. Med., 2011, 50, 1274–1279. ´rez, D. E. Bikiel, D. E. Wetzler, M. A. Martı´ and S. A. Sua F. Doctorovich, Anal. Chem., 2013, 85, 10262–10269. (a) X. Li, X. Gao, W. Shi and H. Ma, Chem. Rev., 2014, 114, 590–659; (b) L. Yuan, W. Lin, K. Zheng, L. He and W. Huang, Chem. Soc. Rev., 2013, 42, 622–661; (c) J.-T. Hou, M.-Y. Wu, K. Li, J. Yang, K.-K. Yu, Y.-M. Xie and X.-Q. Yu, Chem. Commun., 2014, 50, 8640–8643; (d) H. Lv, X.-F. Yang, Y. Zhong, Y. Guo, Z. Li and H. Li, Anal. Chem., 2014, 86, 1800–1807; (e) G. Song, Y. Sun, Y. Liu, X. Wang, M. Chen, F. Miao, W. Zhang, X. Yu and J. Jin, Biomaterials, 2014, 35, 2103–2112; ( f ) L. A. Montoya and M. D. Pluth, Anal. Chem., 2014, 86, 6032–6039; (g) J. Tong, Y. Wang, J. Mei, J. Wang, A. Qin, J. Z. Sun and B. Z. Tang, Chem. – Eur. J., 2014, 20, 4661–4670; (h) C. Huang, T. Jia, M. Tang, Q. Yin, W. Zhu, C. Zhang, Y. Yang, N. Jia, Y. Xu and X. Qian, J. Am. Chem. Soc., 2014, 136, 14237–14244. (a) A. T. Wrobel, T. C. Johnstone, A. D. Liang, S. J. Lippard and P. RiveraFuentes, J. Am. Chem. Soc., 2014, 136, 4697–4705; (b) J. Rosenthal and S. J. Lippard, J. Am. Chem. Soc., 2010, 132, 5536–5537; (c) Y. Zhou, K. Liu, J.-Y. Li, Y. Fang, T.-C. Zhao and C. Yao, Org. Lett., 2011, 13, 1290–1293. (a) C. Liu, H. Wu, Z. Wang, C. Shao, B. Zhu and X. Zhang, Chem. Commun., 2014, 50, 6013–6016; (b) K. Kawai, N. Ieda, K. Aizawa, T. Suzuki, N. Miyata and H. Nakagawa, J. Am. Chem. Soc., 2013, 135, 12690–12696; (c) X. Jing, F. Yu and L. Chen, Chem. Commun., 2014, 50, 14253–14256; (d) G.-J. Mao, X.-B. Zhang, X.-L. Shi, H.-W. Liu, Y.-X. Wu, L.-Y. Zhou, W. Tan and R.-Q. Yu, Chem. Commun., 2014, 50, 5790–5792; (e) Z. Miao, J. A. Reisz, S. M. Mitroka, J. Pan, M. Xian and S. B. King, Bioorg. Med. Chem. Lett., 2015, 25, 16–19. (a) W. Zhang, P. Li, F. Yang, X. Hu, C. Sun, W. Zhang, D. Chen and B. Tang, J. Am. Chem. Soc., 2013, 135, 14956–14959; (b) D. Kim, H. G. Ryu and K. H. Ahn, Org. Biomol. Chem., 2014, 12, 4550–4566; (c) H. Zhang, J. Fan, J. Wang, S. Zhang, B. Dou and X. Peng, J. Am. Chem. Soc., 2013, 135, 11663–11669; (d) H. Yu, Y. Xiao and L. Jin, J. Am. Chem. Soc., 2012, 134, 17486–17489; (e) X.-L. Liu, X.-J. Du, C.-G. Dai and Q.-H. Song, J. Org. Chem., 2014, 79, 9481–9489; ( f ) S. K. Bae, C. H. Heo, D. J. Choi, D. Sen, E.-H. Joe, B. R. Cho and H. M. Kim, J. Am. Chem. Soc., 2013, 135, 9915–9923; (g) K. Zheng, W. Lin, L. Tan, H. Chen and H. Cui, Chem. Sci., 2014, 5, 3439–3448; (h) L. Li, J. Ge, H. Wu, Q.-H. Xu and S. Q. Yao, J. Am. Chem. Soc., 2012, 134, 12157–12167. L. Yuan, W. Lin, H. Chen, S. Zhu and L. He, Angew. Chem., Int. Ed., 2013, 52, 10018–10022.
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Cite this: DOI: 10.1039/c4cc10382c Received 29th December 2014, Accepted 13th February 2015
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A two-photon fluorescent turn-on probe for nitroxyl (HNO) and its bioimaging application in living tissues† Kaibo Zheng,a Weiying Lin,*ab Dan Cheng,a Hua Chen,a Yong Liub and Keyin Liub
DOI: 10.1039/c4cc10382c www.rsc.org/chemcomm
The first two-photon fluorescent probe for specific detection of nitroxyl is designed and synthesized, and we have further demonstrated that the new two-photon fluorescent probe could be employed to image nitroxyl in living cells and tissues.
Nitroxyl (HNO), a one-electron-reduced form of NO, has recently received intense attention due to its biological functions and many potential pharmacological effects in mammalian systems.1 For example, HNO is used clinically as a deterrent for alcohol use, exhibits anti-cancer properties, shows promise for the treatment of heart failure, protects tissue against reperfusion injury, etc.1,2 Thus, it is of high significance to develop efficient methods for the detection of HNO in living systems. To date, several analytical techniques including colorimetric method,1a,3 EPR,4 HPLC,5 mass spectrometry6 and electrochemical analysis1a,7 have been developed for HNO detection. Although these methods provide sensitive analysis, they require complicated sample preparation, sophisticated instrumentation, or destruction of tissues or cells. Therefore, they are not suitable for applications in living systems. In recent years, fluorescent probes, as excellent molecular tools, have attracted increasing attention due to their high selectivity, high sensitivity, as well as real-time imaging, and they have been widely applied in the detection of biological molecules.8 A number of fluorescent probes for HNO based on the HNO-induced reduction of Cu(II) to Cu(I) have been developed.9 Alternatively, by taking advantage of the reaction of HNO with triarylphosphine to give the corresponding phosphine oxide and aza-ylide, fluorescent HNO probes have been engineered.10 However, the fluorescent probes for HNO reported are based on one-photon microscopy (OPM), which only show a shallow penetration depth. Moreover, the short excitation wavelengths a
State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, Hunan 410082, P. R. China b Institute of Fluorescent Probes for Biological Imaging, University of Jinan, Jinan, Shandong 250022, P. R. China. E-mail: [email protected] † Electronic supplementary information (ESI) available: Quantum yields, 1H NMR and 13C NMR data, MTT assay, and UV/vis absorption. See DOI: 10.1039/c4cc10382c
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may lead to photobleaching of probes and damage in cells and tissues, and thus limit their biological applications. By contrast, two-photon microscopy (TPM) is an attractive technique to study biomolecules in living cells and tissues. TPM, where fluorescence is triggered by two-photon excitation, is advantageous over conventional one-photon fluorescence microscopy.11 It can facilitate three-dimensional imaging of living tissues, reduce photodamage to biosamples, increase tissue penetration, and lower background fluorescence. These are favourable for imaging deep tissues. In spite of these advantages, to the best of our knowledge, no two-photon fluorescent HNO probes have been reported in the literature to date. Thus, it is of great interest to design two-photon fluorescent probes, which are suitable for monitoring HNO not only in living cells but also in living tissues. Recently, our group has reported a fluorescent dye GCTPOC, which contains a rigid oxygen-bridge and a hydroxyl group at the 2-position ( para-position). GCTPOC exhibits excellent two-photon properties with a two-photon cross-section (s) above 810 GM and a two-photon excitation action cross-section above 270 GM (excitation at 860 nm), indicating that the dye is potentially useful for bioimaging applications.12 GCTPOC has been demonstrated to be an efficient two-photon platform for designing two-photon probes with its tunable twophoton properties by modifications at the hydroxyl group. In this work, we developed GCTPOC-1 as the first two-photon fluorescent turn-on HNO probe using GCTPOC as the two-photon platform and triarylphosphine as the reactive site for HNO (Scheme 1). The two-photon probe GCTPOC-1 has a large fluorescence enhancement, which makes it attractive for imaging HNO in living tissues with deep tissue penetration. The starting compound GCTPOC was prepared according to a literature procedure.12 All new compounds were characterized by standard NMR spectroscopy and mass spectrometry. With GCTPOC-1 in hand, we examined its optical properties in the absence or presence of HNO (Angeli’s salt (AS) was used as the HNO source in all experiments). The free probe GCTPOC-1 (10 mM) exhibited a maximal absorption band at around 354 nm (e = 5051 M 1 cm 1) in pH 7.4, 25 mM PBS buffer (1% ethanol) at
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Scheme 1 Design and synthesis of the first two-photon fluorescent turn-on HNO probe GCTPOC-1.
ambient temperature (Fig. S1, ESI†). As anticipated, the free GCTPOC-1 showed relatively weak fluorescence (F = 0.001). However, upon treatment with HNO, a drastic enhancement in fluorescence intensity at around 512 nm was observed (Fig. 1). A maximal fluorescence enhancement (49-fold) was obtained when the concentration of AS reached 15 equiv. The detection limit for GCTPOC-1 was calculated to be 5.90 10 7 M (Fig. S2, ESI†), indicating that the probe is highly sensitive to HNO. To confirm the sensing process, we decided to study the product of GCTPOC-1 + HNO by both NMR spectroscopy and mass spectrometry. The product of GCTPOC-1 + HNO was isolated using a silica gel column and was then subjected to 1H NMR analysis. The 1H NMR of the resulting product is essentially identical to that of the authentic GCTPOC (The experiment section of the ESI†). In addition, the identity of the product of GCTPOC-1 + HNO was further characterized by mass spectrometry analysis (Fig. S3, ESI†). This is consistent with the previous report that HNO reacts with triarylphosphines to give the corresponding phosphine oxide.10 The time courses of the fluorescence intensities of GCTPOC-1 (5 mM) in the absence or presence of HNO (5, 10, and 15 equiv.) in pH 7.4, 25 mM PBS buffer (1% ethanol) are displayed in Fig. 2. Notably, a drastic enhancement in fluorescence intensities was observed within a few minutes, and a plateau was reached in 45 minutes in the presence of 5, 10 or 15 equiv. of AS. It suggests that the rapid fluorescence response of the probe may make it suitable for detection of HNO. We then examined the kinetic profiles of the reaction under pseudo-first-order conditions with a large excess of HNO (15 equiv.) over the probe GCTPOC-1
Fig. 1 Fluorescence spectra of GCTPOC-1 (5.0 mM) in pH 7.4 PBS buffer (1% ethanol) in the absence or presence of AS (0–100 mM). Inset: fluorescence intensity ratio (F/F0) changes at 512 nm of GCTPOC-1 (5.0 mM) with the amount of AS. The spectra were recorded after incubation of the probe with AS for 45 min.
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Fig. 2 Reaction-time profiles of GCTPOC-1 (5.0 mM) in the absence [’] or presence of AS (25 mM [ ], 50 mM [ ], and 75 mM [ ]). The fluorescence intensities at 512 nm were continuously monitored at time intervals in pH 7.4 PBS buffer (1% ethanol). Time points represent 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, and 60 min.
(5 mM) in pH 7.4, 25 mM PBS buffer (1% ethanol) at ambient temperature. The pseudo-first-order rate constant for HNO was determined to be k = 0.0487 min 1 (Fig. S4, ESI†). The effect of pH on the fluorescence response of GCTPOC-1 to HNO was investigated. As shown in Fig. S5 (ESI†), in the absence of HNO, only a very minor change in fluorescence intensity was observed in the free probe over a wide pH range of 5.0–8.5, indicating that the free probe was stable in the wide pH range. Upon treatment with AS, the maximal fluorescence signal was observed in the pH range of 7.0–8.5, indicating that the probe can be employed to detect HNO at a physiological pH of 7.4. However, the probe has relatively low sensitivity at lower pH. This may be improved in the future. The probe GCTPOC-1 was treated with various relevant analytes including anions, reactive oxygen species, reducing agents, small molecule thiols, and AS in pH 7.4, 25 mM PBS buffer (1% ethanol) to investigate the selectivity. As shown in Fig. 3, the addition of the representative species (ClO , H2O2, Fe3+, O2 , sodium ascorbate, N3 , NO2 , NO3 , Cys, Na2S (H2S), CORM-2 (CO), NO at 100 mM, and GSH at 1 mM) elicited essentially no fluorescence response for over 45 min. By contrast, importantly, introduction of AS (75 mM) caused a large fluorescence enhancement (49-fold) in 45 min. Notably, S-nitrosoglutathione (GSNO) at 100 mM triggered only a small fluorescence enhancement (o9 fold) and had nearly no interference for HNO detection. These data indicate that the probe is selective for HNO over other tested species. It is necessary to evaluate both the photostability and chemical stability of fluorescent probes designed to be used in biological environments. The photostability of GCTPOC-1 in pH 7.4, 25 mM PBS buffer (1% ethanol) at ambient temperature was measured by continuous irradiation with UV light (365 nm), and the results demonstrate that the emission profile of GCTPOC-1 has only minor changes after 1 h of irradiation (Fig. S6A, ESI†), indicating that this probe has sufficient photostability for potential biological use. In the experimental procedure, AS (the HNO source) was dissolved in 0.01 M NaOH solution in order to investigate
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Fig. 3 The fluorescence responses of the probe GCTPOC-1 (5.0 mM) to various relevant species (100 mM): 1. Blank; 2. ClO ; 3. H2O2; 4. sodium ascorbate; 5. O2 ; 6. NO; 7. NO2 ; 8. NO3 , 9. N3 ; 10. Fe3+; 11. CO; 12. H2S; 13. Cys; 14. GSH; 15. GSNO; and 16. AS, in pH 7.4, 25 mM PBS buffer (1% ethanol).
whether the NaOH solution (225 mL, 0.01 M NaOH) hydrolyzes the probe in pH 7.4, 25 mM PBS buffer (1% ethanol). There are many oxidizing and reducing reagents in biological settings. Thus, we further investigated the emission profiles of the probe GCTPOC-1 in the presence of the representative NaOH, oxidizing and reducing reagents. As a representative case, GCTPOC-1 in pH 7.4, 25 mM PBS buffer (1% ethanol) at ambient temperature was treated with H2O2, NaClO, Fe3+, Cys at 100 mM, NaOH at 0.75 mM or GSH at 1 mM for 1 h. As shown in Fig. S6B (ESI†), the emission profile of GCTPOC-1 is almost unchanged. Thus, these data suggest that the probe GCTPOC-1 displays sufficient chemical stability. In order to be useful as imaging agents, fluorescent probes should have low cytotoxicity. Thus, we investigated the potential toxicities of GCTPOC-1 against a representative cell line, HeLa cells. The living cells were incubated with various concentrations (5–50 mM) of GCTPOC-1 for 24 h, and then the cell viability was determined by the standard 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assays. The results indicate that GCTPOC-1 has no marked cytotoxicity at concentrations below 30 mM (Fig. S7, ESI†). Thus, the prominent features of GCTPOC-1 include good two-photon fluorescence properties, high sensitivity, excellent selectivity, relatively fast response, good functioning at physiological pH, and low cytotoxicity. These desirable attributes prompted us to further examine the suitability of the probe for visualizing HNO in living systems. Toward this end, the probe was incubated with HeLa cells in the absence or presence of AS, and the fluorescence images were recorded both in one-photon and two-photon modes. In the one-photon mode, the HeLa cells incubated with only the probe GCTPOC-1 (5.0 mM) for 20 min at 37 1C in PBS exhibited very weak fluorescence at the emission window of 470–570 nm (Fig. 4b). By sharp contrast, HeLa cells incubated with 5.0 mM GCTPOC-1 for 20 min, and then treated with AS (35 mM, 75 mM) displayed strong fluorescence (Fig. 4f and j). Consistently, in the two-photon mode (excitation at 780 nm), the HeLa cells incubated with only the probe showed almost no fluorescence (Fig. 4d). However, the cells pretreated with GCTPOC-1 and
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Fig. 4 One-photon fluorescent images: (a) bright-field image of living HeLa cells incubated with only GCTPOC-1 (5.0 mM) for 20 min; (b) Fluorescence image of (a); (e, i) bright-field image of living HeLa cells incubated with GCTPOC-1 (5.0 mM) for 20 min, then with AS (35 mM or 75 mM) for 45 min; (f, j) Fluorescence image of (e, i). Excitation at 405 nm. Two-photon fluorescence images: (c) bright-field image of living HeLa cells incubated with only GCTPOC-1 (5.0 mM) for 20 min; (d) fluorescence image of (c); (g, k) bright-field image of living HeLa cells incubated with GCTPOC-1 (5.0 mM) for 20 min, then with AS (35 mM or 75 mM) for 45 min; (h, l) fluorescence image of (g, k). Excitation at 780 nm. Scale bar = 20 mm.
further incubated with the AS (35 mM, 75 mM) exhibited bright fluorescence (Fig. 4h and l). The comparison among Fig. 4b, f, and j (Fig. 4d, h, and l) reveals a dose-dependent fluorescence enhancement. Thus, these data establish that the probe GCTPOC-1 is cell membrane permeable and capable of sensing HNO in living cells. When compared to the traditional one-photon fluorescence microscopy with emission in the visible region, the major advantage of two-photon fluorescence microscopy is that the latter is suitable for three-dimensional fluorescence imaging in thick living samples, for instance, living tissues, due to the localized excited volume. We were interested in exploiting the capability of the probe GCTPOC-1 in this aspect. Toward this end, living tissue slices (a thickness of 400 mm) of the liver were prepared and subjected to one-photon and two-photon fluorescence microscopic analyses. In a control experiment, the tissue slices incubated with only GCTPOC-1 (30 mM) for 40 min at 37 1C in PBS exhibited weak fluorescence in one-photon (Fig. S8, ESI†) and two-photon fluorescence modes (Fig. S9, ESI†) at the emission window of 520–570 nm. By contrast, when the tissue slices were incubated with 30 mM GCTPOC-1 for 40 min, and then treated with 1 mM AS, relatively significant fluorescence is only observed up to 70 mm in the one-photon fluorescence mode (Fig. S10, ESI†). However, in the two-photon mode (excitation at 780 nm), relatively significant fluorescence is observed up to 130 mm (Fig. 5). These data indicate that the probe GCTPOC-1 is capable of detecting HNO at 130 mm depth in living tissues using two-photon fluorescence microscopy, in accord with its good two-photon fluorescence properties. In summary, we have constructed the first two-photon fluorescent turn-on HNO probe GCTPOC-1 based on a two-photon platform with a large cross-section, GCTPOC, and a sensitive HNO
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Fig. 5 Two-photon fluorescence images of a fresh rat liver slice pretreated with 30 mM GCTPOC-1 and then with AS (1 mM) incubated at depths of approximately 0 B 150 mm with a magnification of 10. Excitation at 780 nm. Scale bar = 200 mm.
recognition site. The probe GCTPOC-1 exhibits desirable properties including high sensitivity, high selectivity, good functioning at physiological pH and low cytotoxicity. In particular, the probe shows a 49-fold enhancement in the presence of AS (75 mM). Importantly, we have demonstrated that the probe GCTPOC-1 is suitable for fluorescence imaging of HNO not only in living cells, but also in living tissues using two-photon fluorescence microscopy. Further applications of the two-photon probe for the investigation of the biological functions and pathological roles of HNO in living systems are under progress. This work was financially supported by the NSFC (21172063, 21472067) and the startup fund of University of Jinan.
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