Chinese Chemical Letters  2014, Vol.25 Issue (01):19-23   PDF    
引用本文
Qing Liu, Lin Xue, Dong-Jian Zhu, Guo-Ping Li, Hua Jiang.Highly selective two-photon fluorescent probe for imaging of nitric oxide in living cells[J]. Chin. Chem. Lett., 2014,25(01): 19-23   
Highly selective two-photon fluorescent probe for imaging of nitric oxide in living cells
Qing Liua,c, Lin Xuea, Dong-Jian Zhua,c, Guo-Ping Lia,c, Hua Jianga,b     
* Corresponding authors at:a Beijing National Laboratory for Molecular Sciences, CAS Key Laboratory of Photochemistry, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China;
b College of Chemistry, Beijing Normal University, Beijing 100875, China;
c University of Chinese Academy of Sciences, Beijing 100049, China
Received:2013-10-12, Revised:2013-11-8, online:2013-11-20.
E-mail addresses:liguoping08@iccas.ac.cn;hjiang@iccas.ac.cn
Abstract: A new two-photon fluorescent probe, ADNO, for nitric oxide (NO) based on intramolecular photoinduced electron transfer (PET) mechanism displays a rapid response to NO with a remarkable fluorescent enhancement in PBS buffer. The excellent chemoselectivity of ADNO for NO over other ROS/RNS (reactive oxygen species or nitrogen species) and common metal ions was observed. Moreover, ADNO has been successfully applied in fluorescence imaging of NO of living cells using both one-photon microscopy (OPM) and two-photon microscopy (TPM).
Key words: Fluorescent probe     Nitric oxide     Fluorescence imaging     One-photon microscopy     Two-photon microscopy    
1. Introduction

Nitric oxide (NO), an important and ubiquitous endogenous second messenger or cytostatic/cytotoxic agent, has attracted more and more attention in recent years [1]. It is believed that NO is, at low concentrations, essential for many normal physiological processes and plays an important role in several physiological functions, such as regulation of vascular tone, neurotransmission, anti-viral defense, immune response and memory. Whereas excessive generation of NO in physiological processes can cause damage to biomolecules, such as DNA, proteins and lipids, and consequently lead to cellular necrosis or apoptosis. Therefore, sensitive and specific detection of NO in living systems is indeed indispensable for understanding the biological roles of NO. The development of techniques to analyze NO in organisms has dramatically increased over the last decades [2], especially, fluorescence microscopy, which features non-destruction, high sensitivity and spatiotemporal resolution, has been proved to be a central tool for visualizing various ROS/RNS (reactive oxygen species or nitrogen species) [3]. Compared with standard laser confocal approaches which employ one photon of higher energy for the excitation, two-photon microscopy of lower energy as the excitation source has significant advantages by providing deeper sectioning, less phototoxicity, and lower background fluorescence [4]. Although a number of NO-specific fluorescent probes have been reported to date [5,3b,c,e], two-photon fluorescent probes for imaging of NO suitable for biological applications are still rare.

In this report, we designed and synthesized a new two-photon fluorescent probe, ADNO (2-(α-(3,4-diaminophenoxy)acetyl)-6- (dimethylamino)naphthalene), for monitoring NO based on photoinduced electron transfer (PET) mechanism. As shown in Scheme 1, the probe is composed of two moieties: o-phenylenediamine as the NO-sensitive fluorescence modulator and 2-acetyl- 6-(dimethylamino)naphthalene (Acedan) as the two-photon fluorophore. We anticipated that detection of NO can be well achieved by altering the electron-donating capacity of the ophenylenediamine moiety. The electron-rich o-phenylenediamine group should quench the fluorescence of Acedan due to the existence of electron transfer from electron-rich diamine moiety to the excited fluorophore. However, the presence of NO under aerobic conditions leads to the transformation of o-phenylenediamine to benzotriazole, which consequently suppresses the PET process and revives the strong fluorescence response of the probe.

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Scheme 1.Design strategy of probe ADNO.
2. Experimental

All chemicals were purchased from Alfa Aesar or Sigma-Aldrich and used as received. All solvents were purified and dried by standard methods prior to use. Pure water (18.2 Ω) was used to prepare all aqueous solutions. Various reactive oxygen species were prepared according to the reported literature [6]. The 1HNMR spectra were recorded on a Bruker AVANCE-400 spectrometer, and 13C NMR spectra were recorded on a Bruker AVANCE-600 MHz spectrometer. All chemical shifts are reported in the standard notation of parts per million using residual solvent protons as internal standard. Mass spectra (ESI) were obtained on Bruker Apex IV FTMS. UV-vis spectra were recorded on SHIMADZU UV- 2550 UV-vis spectrometer. Fluorescence spectra were recorded using a HITACHI F-4600 spectrometer. OPM imaging experiments were performed with an Olympus FV-1000 laser scanning microscopy system, based on an IX81 (Olympus, Japan) inverted microscope. TPM imaging experiments were performed with an Olympus FV1000MPE two-photon excitation fluorescence microscopy system. The quantum yields for fluorescence were calculated by comparison of the integrated area of the corrected emission spectrum of the samples with that of a solution of quinine sulfate in 0.1 mol/L H2SO4 (Φ= 0.54) [7].

NIH 3T3 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal calf serum (FCS, Gibco), 50 μg/mL penicillin/streptomycin (Hyclone) at 37 ℃ in a humidified incubator containing 5% CO2 gas. The cells were plated in a 35 mm glass-bottomed dish and cultured for 2 days before dye loading. Then the cells were washed with phosphatebuffered saline (PBS) and bathed in corresponding serum-free DMEM medium with 4 μmol/L ADNO for 20 min at 25 ℃, washed with PBS three times to remove the excess probe and bathed in PBS (2 mL) before imaging.

The synthetic procedures are outlined in Scheme 2. The compounds 1 and 2 were synthesized according to previously published methods [8]. Compound 3 can be easily obtained in a good yield by coupling of the compound 2 with 4-amino-3- nitrophenol under basic conditions. Then reduction of compound 3 with sodium dithionite gave the final product ADNO.

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Scheme 2.Synthetic procedure for probe ADNO.
3. Results and discussion

To evaluate whether our probe can monitor NO, we first examined the spectral properties of ADNO in PBS buffer (20% DMSO was added as a co-solvent). As predicted, ADNO only displayed weak fluorescence emission (ε = 1.31 × 104 L mol-1 cm at 386 nm, Φ0 = 0.01) owing to the PET process between ophenylenediamine group and fluorophore Acedan (Fig. 1a). Addition of NO (2 mmol/L saturated solution in oxygen-free water) to ADNO in PBS buffer induced a rapid and remarkable enhancement on fluorescence intensity (ε = 1.79 × 104 L mol-1 cm-1 at 356 nm, Φ = 0.08,Φ/Φ0 = 8). Meanwhile, the absorption spectrum of ADNO centered at 390 nm also blue-shifted to 358 nm (Fig. S1 in Supporting information). It can be well explained that the formation of benzotriazole (Fig. S2 in Supporting information) mediated by NO under aerobic conditions has suppressed the PET process. Considering that NO has been discovered to circulate as an S-nitroso adduct of serum albumin, [9] we then applied the probe to monitor the process of S-nitrosocysteine (SNOC, a NO donor) spontaneously releasing NO in aqueous solution (Fig. 1b). After 100 μmol/L SNOC was added, the fluorescence intensity increased gradually and reached the same level as NO solution after 1 h. These results clearly indicate that ADNO can rapidly respond to NO and monitor the generation and accumulation of NO in aqueous solution.

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Fig. 1.(a) Fluorescence emission spectra of 4 μmol/L ADNO upon addition of 40 μmol/L NO. (b) Fluorescence emission spectra of 4 μmol/L ADNO upon addition of 100 μmol/L SNOC changing with time (every 5 min). Spectra were acquired in PBS buffer (20% DMSO, pH 7.40), λex = 405 nm, λem = 546 nm.
Next, we investigated the chemoselectivity of ADNO for NO by fluorimetric titration of various ROS/RNS and common metal ions in the organism. Only the NO solution and NO donor can induce remarkable fluorescence response (Fig. 2). Other ROS/RNS, including singlet oxygen (1O2), superoxide (O2 -), hydroxyl radical (·OH), hypochlorite (ClO-), hydrogen peroxide (H2O2), nitrite (NO2-) and nitrate (NO3-), gave out no obvious changes in fluorescence emission intensity. Common metal ions in organisms, such as Na+, K+, Ca2+, Mg2+, Zn2+, Fe2+, Cu2+, Co2+, Ni2+, had no effect on the fluorescence emission of ADNO as well (Fig. S3 in Supporting information). Therefore, we conclude that the probe ADNO has excellent chemoselectivity for NO over other competing ROS/RNS and common metal ions in PBS buffer.

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Fig. 2.Fluorescence responses of ADNO (4 μmol/L) to NO (40 μmol/L), SNOC (100 μmol/L) and other ROS/RNS (200 μmol/L). All the data were acquired 5 min after addition of each ROS/RNS except for SNOC (1 h) in PBS buffer (20% DMSO, pH 7.40). λex = 405 nm, λem = 546 nm.
In addition,we also assessed the NO-sensing ability of ADNO at different pH values. As shown in Fig. 3, probe ADNO is stable and showsweak fluorescencewithin awide pH range from 6.5 to 10.5. The change of pH did not obviously affect the fluorescence enhancement. Though maximum intensity is achieved at about pH > 7.5, the change of fluorescence intensity at pH 6.5-7.5 is still adequate enough for the signal output in sensing NO. These findings suggested that probeADNOis suitable for the detection of NO within biological pH range.

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Fig. 3.Fluorescence responses of ADNO (4 μmol/L) to NO (40 μmol/L) in PBS buffer (20% DMSO, pH 7.40) at various pH values. F/F0 means the ratio (▲) between fluorescence intensity of ADNO in the absence (■, F0) and presence of NO (●, F), λex = 405 nm, λem = 546 nm.
With these data in hand, we further examined the bioimaging applications of ADNO for NO in living cells. NIH 3T3 cells were incubated with 4 μmol/L of ADNO for 20 min at 25 ℃ and then washed three times with PBS buffer to remove the remaining dyes. The ADNO-labeled cells exhibit only faint fluorescence in the optical window 460-560 nm by one-photon microscopy (OPM) (Fig. 4b). Just as the phenomena found in aqueous media (Fig. 1b), the fluorescence emission gradually increased after 5 mmol/L SNOC (NO donor) was added, and moderate fluorescence enhancement was achieved after 30 min (Fig. 4d). Under the guide of OPM imaging experiments, the TPM imaging experiments were also carried out (Fig. 5). As we expected, similar results were observed by two-photon microscopy excited at 820 nm with fs pulses. The gradually enhanced fluorescence emission clearly indicated the generation and accumulation of NO in NIH 3T3 cells released by SNOC. Thus, we can conclude that ADNO has been successfully used for bioimaging of NO in living cells by both OPM and TPM.

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Fig. 4.OPM images of NO in ADNO-labeled NIH 3T3 cells. (a) Bright-field image of cells stained with 4 μmol/L ADNO at 25 ℃ for 20 min. (b) OPM images of (a). (c)OPM images of ADNO-labeled cells treated with 5 mmol/L SNOC for 15 min and (d) 30 min at 25 ℃. Emission intensities were collected in an optical window 460–560 nm, λex = 405 nm, Scale bar: 30 μ；m, intensity bar: 100–3000.

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Fig. 5.TPM images of NO in ADNO-labeled NIH 3T3 cells. (a) Bright-field image of cells stained with 4 μmol/L ADNO at 25 ℃ for 20 min. (b) TPM images of (a). (c) TPM images of ADNO-labeled cells treated with 5 mmol/L SNOC for 15 min and (d) 30 min at 25 ℃. Emission intensities were collected in an optical window 520–560 nm, λex = 820 nm, Scale bar: 30 μm, intensity bar: 100–2000.
4. Conclusion

In summary, on the basis of PET mechanism, we present a new two-photon fluorescent probe for detection of NO. The probe ADNO can rapidly respond to NO with significant turn-on fluorescence, high selectivity, and less pH-dependency. Moreover, we have successfully applied ADNO to monitor the generation and accumulation of NO in living cells by both OPM and TPM. We also expect that this new two-photon fluorescent probewill be a practical tool for NO-related biological research.

Acknowledgments

We thank the National Natural Science Foundation of China (Nos. 21102148 and 21125205), National Basic Research Program of China (No. 2011CB935800), and the State Key Laboratory of Fine Chemicals, Department of Chemical Engineering, Dalian University of Technology for financial supports.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cclet.2013.11.024.

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