A Highly Stable Two-Photon Ratiometric Fluorescence Probe for Real-Time Biosensing and Imaging of Nitric Oxide in Brain Tissues and Larval Zebrafish

Abstract

Open AccessCCS ChemistryRESEARCH ARTICLE6 Jun 2022A Highly Stable Two-Photon Ratiometric Fluorescence Probe for Real-Time Biosensing and Imaging of Nitric Oxide in Brain Tissues and Larval Zebrafish Zhiwen Gong, Zhichao Liu, Zhonghui Zhang, Yuxiao Mei and Yang Tian Zhiwen Gong Shanghai Key Laboratory of Green Chemistry and Chemical Processes, School of Chemistry and Molecular Engineering, East China Normal University, Shanghai 200241 Google Scholar More articles by this author , Zhichao Liu Shanghai Key Laboratory of Green Chemistry and Chemical Processes, School of Chemistry and Molecular Engineering, East China Normal University, Shanghai 200241 Google Scholar More articles by this author , Zhonghui Zhang Shanghai Key Laboratory of Green Chemistry and Chemical Processes, School of Chemistry and Molecular Engineering, East China Normal University, Shanghai 200241 Google Scholar More articles by this author , Yuxiao Mei Shanghai Key Laboratory of Green Chemistry and Chemical Processes, School of Chemistry and Molecular Engineering, East China Normal University, Shanghai 200241 Google Scholar More articles by this author and Yang Tian *Corresponding author: E-mail Address: [email protected] Shanghai Key Laboratory of Green Chemistry and Chemical Processes, School of Chemistry and Molecular Engineering, East China Normal University, Shanghai 200241 Google Scholar More articles by this author https://doi.org/10.31635/ccschem.021.202101038 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail Because nitric oxide (NO) plays important roles in nerve conduction, signal regulation, and immune protection, analysis of NO is of great significance for understanding the physiological and pathological processes related to neurological diseases. Herein, a highly stable and selective two-photon ratiometric fluorescent probe was developed for real-time sensing and imaging of NO in neurons, brain tissues, and larval zebrafish, in which a Rhodamine B derivative (RBD) was designed for specific recognition of NO and gold nanoclusters (AuNCs) were synthesized as reference element. The developed organic–inorganic nanoprobe exhibited high stability against biological thiol compounds and high selectivity against other reactive oxygen and nitrogen species, metal ions, and acids. In addition, the response time of the present nanoprobe was less than ∼55 s. By using the developed nanoprobe, we proved that hypoxia-induced neuronal death was regulated by NO. Moreover, it was found that the hypoxia-induced NO increase in different brain regions was various and that the NO burst contributed to hypoxia-induced death of zebrafish. Download figure Download PowerPoint Introduction Nitric oxide (NO) is an important signal molecule in living systems and plays important roles in nerve conduction, signal regulation, and immune protection.1–3 The disorder of NO concentration has a close relationship with physiological and pathological processes, especially in the brain, including Parkinson’s disease, Alzheimer’s disease, focal cerebral ischemia, and stroke.4–9 Since NO has high affinity for interaction with other biological species, such as oxygen (O2), superoxide anion and peroxides,10 and the concentration of NO changes dynamically in living organisms, it is challenging to monitor the fluctuations of NO in real time in living organisms with high selectivity and accuracy. So far, there are many elegant methods for determination of NO, such as electron paramagnetic resonance spectroscopy, electrochemical methods, Raman spectroscopy, and fluorescent methods.11–14 Our group is very interested in the development of analytical methods for biosensing of biological substances in the brain, including reactive oxygen and nitrogen species (ROS/RNS),15–18 proteins,19,20 and metal ions.21–27 Especially, we have developed a trisoctahedral gold nanostructures-based Raman probe for selectively biosensing NO in live cells,14 but it is limited in real-time imaging of NO in living cells because Raman mapping of cells takes a long time. Fluorescent methods have attracted great attention because of their unique advantages in noninvasive and real-time sensing and imaging of biological samples.28–31 The fluorescent probes currently used for NO detection are mostly organic fluorescent molecules based on o-phenylenediamine,32 which usually suffer from poor water solubility and photobleaching.32,33 It is essential to develop a fluorescence analytical method for real-time bioimaging and quantification of NO in living cells with high stability and selectivity. In this work, a ratiometric two-photon fluorescence (TPF) probe was designed for real-time imaging and biosensing of NO in neurons, brain tissues, and larval zebrafish, in which a Rhodamine B derivative (RBD) was designed for specific recognition of NO, and gold nanoclusters (AuNCs) were synthesized as the reference element (Scheme 1). The developed organic–inorganic nanoprobe showed two clearly separated TPF emissions around ∼540 and ∼580 nm under the excitation of 800 nm. The present nanoprobe displayed good water solubility and high stability against biological sulfhydryl compounds and rapid response time (∼55 s) toward NO. In addition, it displayed a wide detection range of 0.5–120 μM for NO, as well as high selectivity for NO against other ROS/RNS, metal ions, and amino acids. This developed nanoprobe, was applied for real-time imaging of NO in neurons, and it was found that hypoxia-induced neuronal death was regulated by NO. Taking advantage of two-photon imaging with deep penetration, we found that the hypoxia-induced NO increase in different brain regions was different, and the NO burst contributed to hypoxia-induced death of larval zebrafish. Scheme 1 | (a) Synthesis route for RBD. (b) Working mechanism of [email protected] probe for determination of NO. Download figure Download PowerPoint Experimental Methods Synthesis of RBD probe Compound 1 Triethylamine (8.34 mL, 59.71 mmol), 4-iodo-1,2-diaminobenzene (4 g, 17.1 mmol) and (trimethylsilyl) acetylene (4.17 mL, 29.92 mmol) were added into anhydrous tetrahydrofuran (THF; 25 mL). Then N2 was bubbled for a period of time to remove the oxygen of the reaction mixture. Next, CuI (0.16 g, 0.85 mmol) and Pd(PPh3)4 (750 mg, 0.65 mmol) were added to the reaction during the N2 bubbling. The reaction was allowed to take place overnight, and the gray solid product obtained with yield was 85% by purification (ethyl acetate:petroleum ether = 1:4, v/v). Proton nuclear magnetic resonance (1H NMR) [dimethyl sulfoxide (DMSO)-d6, δ]: 6.59 (s, 1H), 6.53–6.48 (m, 1H), 6.45–6.40 (m, 1H), 4.89 (s, 2H), 4.56 (s, 2H), 0.18 (s, 9H). Carbon nuclear magnetic resonance (13C NMR) (DMSO-d6, δ): 137.13, 134.86, 122.16, 117.57, 114.09, 109.96, 108.64, 89.73. Compound 2 The obtained compound 1 (3 g, 14.54 mmol) and K2CO3 (5 g, 36.18 mmol) were added to a mixed solution of MeOH and CH2Cl2 (1:1, v/v), and stirred at 25 °C for 5 h. After the reaction was done, saturated brine (45 mL) was added to the postreaction system, and then extracted with CH2Cl2 (80 mL). Saturated brine was further poured into the final organic phase and dried with sodium sulfate and then rotary-evaporated to a small volume. Finally, it was further purified to obtain an oily product (CH2Cl2:n-hexane = 7:3, v/v) with a yield of 80% (1.54 g, 11.63 mmol). 1H NMR (MeOD, δ): 6.81 (d, J = 1.8 Hz, 1H), 6.74 (dd, J = 8.0, 1.8 Hz, 2H), 6.61 (d, J = 8.0 Hz, 1H), 3.16 (s, 1H). 13C NMR (MeOD, δ): 136.27, 133.96, 123.61, 119.29, 115.16, 111.97, 84.54, 73.76. Compound RBD Through a constant pressure funnel, SOCl2 (0.11 g, 0.9 mmol) was gradually added dropwise to Rhodamine B (0.14 g, 0.30 mmol) in dichloromethane (20 mL) at 25 °C. After the device was cooled, the solvent was spin-dried to obtain Rhodamine B acid chloride. Then, Rhodamine B acid chloride and tetraethylammonium (TEA; 8 mL) were added to anhydrous acetonitrile (20 mL), and compound 2 (0.21 g, 1.57 mmol) dissolved in anhydrous acetonitrile (20 mL) was added dropwise to the above mixture. After stirring for 4 h at 25 °C, the mixture was concentrated in vacuo to a small volume and then further purified (CH2Cl2:MeOH = 80:1, v/v). A yellow-white powder was obtained with yield of 60%. 1H NMR (CDCl3, δ): 8.05 (d, J = 7.1 Hz, 1H), 7.60 (dd, J = 17.8, 9.6 Hz, 2H), 7.33 (d, J = 7.0 Hz, 1H), 7.10 (d, J = 11.6 Hz, 1H), 6.63 (dd, J = 13.2, 8.9 Hz, 2H), 6.46 (d, J = 8.3 Hz, 1H), 6.41–6.26 (m, 4H), 6.11–6.05 (m, 1H), 3.56 (s, 2H), 3.42–3.28 (m, 8H), 2.72 (s, 1H), 1.17 (t, J = 7.0 Hz, 12H). 13C NMR (CDCl3, δ): 166.23, 153.83, 149.07, 145.54, 144.33, 132.81, 132.21, 128.67, 128.63, 128.56, 128.42, 124.43, 120.53, 116.16, 111.06, 108.00, 106.62, 97.98, 83.88, 77.28, 77.03, 76.78, 74.18, 68.21, 44.45, 12.52. Synthesis of [email protected] nanoprobe Bovine serum albumin (BSA)-stabilized AuNCs were synthesized according to the method previously reported in the literature.34 The mixture of HAuCl4 (4 mL, 10 mM) and BSA solution (4 mL, 20 mg/mL) was vigorously stirred at 37 °C for 3 min, and 40 μL of AA (0.35 mg/mL) was added to the mixture, followed by addition of an appropriate amount of NaOH (1 M) under stirring to control the pH of the mixed solution that reached ∼8. The mixture was further stirred at 37 °C for 5 h. AuNCs were obtained by centrifugation at 10,000 rpm for 15 min. Then, RBD solution (1 mL, 10 mM) was bubbled with N2 for half an hour to remove dissolved oxygen, followed by addition of AuNCs, and the mixture reacted at 60 °C for 6 h. Excess RBD was removed by centrifugation treatment (6000 rpm, 3 min). The produced [email protected] probe was resuspended in phosphate-buffered saline (PBS) buffer before being used. Confocal fluorescence imaging All animal experiment protocols strictly complied with the implementation guidelines of Care and Use of Laboratory Animals formulated by the Ministry of Science and Technology of China and have been approved by the Animal Protection and Use Committee of East China Normal University (approval number: m+R20190304, Shanghai, China). To image NO in living cells, neurons were first incubated with the [email protected] probe in Hank’s balanced salt solution (HBSS) buffer for 30 min. Then, the neurons were washed three times by PBS to remove unabsorbed probe. Next, different groups of neurons were stimulated by different concentrations of NO and further used for confocal imaging. Similar experiments were also conducted under hypoxia treatment for different times before imaging. For the control experiment, the neurons were first incubated with the [email protected] probe and 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide potassium (Carboxy-PTIO) for 30 min, and then the neurons were washed three times by PBS to remove unabsorbed probe and Carboxy-PTIO. Subsequently, the neurons were stimulated by hypoxia for a different time before imaging. For confocal fluorescence imaging of NO in brain slices, different brain slices were obtained from 2-month-old mice treated by decapitation. Then, the brain slices were transferred into artificial cerebrospinal fluid (aCSF) buffer and further incubated with the [email protected] probe for 30 min. Next, the brain slices were washed three times by aCSF to remove unabsorbed probe before imaging. Similar experiments were also conducted under hypoxia treatment for different times before imaging. For confocal fluorescence imaging of NO in larval zebrafish with the stimulation of hypoxia, 3-day-old larval zebrafish were first incubated with [email protected] probe in nutrient solution for 60 min. Then the larval zebrafish were washed three times by nutrient solution to remove unabsorbed probe. Next, N2 was continuously blown into the nutrient solution to remove O2 before imaging. Results and Discussion Design, synthesis, and characterization of [email protected] probe For specific identification of NO, an RBD was designed in three parts: the first part—Rhodamine B as fluorophore, the second part—o-phenylenediamine as recognition site, and the third part—an alkynyl group as anchor group (Scheme 1a). RBD was synthesized and characterized by NMR and mass spectrometry (MS) ( Supporting Information Figures S1–S9). Because a lactam ring in RBD was formed through the reaction between the lactone group and o-phenylenediamine, resulting in the destruction of the conjugate structure of Rhodamine B, absorption was observed around 550 nm for RBD (Figure 1a). However, the diazotization of the amino group induced by the reaction between NO and RBD resulted in the spiral ring opening of RBD (Scheme 1b). Thus, the fluorescence of RBD was recovered, with typical absorption at ∼556 nm and fluorescence emission around 580 nm (Figure 1a). In addition, NO induced an obvious increase of TPF of RBD, with two-photon cross-section values (δ) increased from 10 ± 3 GM to 103 ± 5 GM under the excitation of 800 nm (Rhodamine 6G in methanol as standard, 1 GM = 1 × 10−50 cm4 s) (Figure 1b). Figure 1 | (a) UV–vis absorption spectra and fluorescence emission spectra of RBD in the absence (1 and 2) and in the presence of NO (3 and 4). (b) Two-photon action spectra of RBD in the absence (1) and in the presence of NO (2) (Error bars: S.D., n = 3). (c) TEM image of AuNCs. Inset shows the particle size distribution of AuNCs. (d) FT-IR characterization of [email protected] after exposure to 1 mM thiols (GSH, Cys, and Hcy) for 2 h. Download figure Download PowerPoint To develop a ratiometric fluorescence probe with good water solubility and internal reference, BSA-templated AuNCs were synthesized according to the previously reported method.32 Transmission electron microscopy (TEM) results showed that the synthesized AuNCs were nondispersed with an average size of 1.5 ± 0.3 nm (Figure 1c), and X-ray photoelectron spectroscopy (XPS) spectra of Au 4f region confirmed two typical binding energies of Au(0) at 4f7/2 (84.8 eV) and 4f5/2 (88.5 eV) electrons in AuNCs ( Supporting Information Figure S10d). Moreover, the synthesized AuNCs displayed a broad absorption and apparent TPF emission around 540 nm ( Supporting Information Figure S10). Then, a ratiometric TPF probe was assembled by conjugating RBD onto AuNCs through a robust Au–C≡C bond,35 named as [email protected] The Fourier transform infrared (FT-IR) spectrum of [email protected] exhibited the disappearance of typical vibration peaks of C≡C (3293 cm−1) and ≡C–H (2098 cm−1) ( Supporting Information Figure S11a), strongly proving the successful conjugation of RBD onto AuNCs through the Au–C≡C bond. Meanwhile, the contact angle of [email protected] was 11.4 ± 1.8° (n = 5, S.D.), which was much smaller than that of RBD (73.0 ± 2.0°, n = 5, S.D.) ( Supporting Information Figure S11b), demonstrating that the developed [email protected] showed better water solubility than that of individual RBDs and benefited from the good hydrophility of BSA-templated AuNCs. Since there are many biological sulfhydryl compounds in live cells, the stability of the developed [email protected] probe exposed to a high concentration (1 mM) of glutathione (GSH), cysteine (Cys), and homocysteine (Hcy) for 2 h was further tested. As shown in Figure 1d, no signal ascribed to the alkynyl group was observed from the FT-IR spectra, demonstrating the high stability of the [email protected] probe, which can be attributed to the highly stable Au–C≡C bond. Analytical performance of [email protected] probe for determination of NO To confirm the working principle, [email protected] were further used for the determination of NO in cell lysis. As illustrated in Figure 2a, the individual [email protected] probe showed single TPF emission around 450 nm under the excitation of 800 nm. With the addition of NO from 0 to 132 μM, an obvious increase for TPF emission at ∼580 nm (Fred: 550–600 nm) was observed, while change was seldom observed for the TPF intensity around 450 nm (Fgreen: 430–500 nm), leading to a ratiometric response to NO. The intensity ratio of Fred to Fgreen (Fred/Fgreen) displayed a good linear relationship with NO concentrations in the range of 0.5–120.0 μM (Figure 2b), which was better or comparable with previously reported methods ( Supporting Information Table S2), and the detection limit was estimated to 105.0 ± 3.1 nM (S/N = 3, n = 20). In comparison with the fluorescent NO probes in the literature,36–42 our developed [email protected] probe showed a wider detection linear range. It should be noted that the absorbance intensity of individual RBD apparently increased around 556 nm with the increasing concentrations of NO, accompanied with fluorescence enhancement around 580 nm ( Supporting Information Figure S12). Thus, the increase of TPF emission at ∼580 nm can be attributed to the production of Rhodamine B, which was further confirmed by MS ( Supporting Information Figure S13). In addition, the response time of developed [email protected] toward NO (100 μM) was evaluated to ∼55 s (Figure 2c), which was faster than most reported fluorescence methods for NO sensing,36–42 as summarized in Supporting Information Table S2. Moreover, the signal variation for [email protected] was <3.7% at pH 6.5–8.0, and little signal decrease (<4.2%) was found for [email protected] when exposed to a Xe lamp (90 W) for 150 min ( Supporting Information Figure S14), indicating the high stability of the designed [email protected] nanoprobe. Figure 2 | (a) TPF emission spectra of [email protected] probe with the addition of different concentrations of NO (0, 0.5, 20, 30, 40, 50, 60, 70, 80, 100, 120, 125, and 132 μM). (b) Working calibration plot between Fred/Fgreen (Fgreen: 430–500 nm, Fred: 550–600 nm) of [email protected] probe and NO concentrations (n = 20, S.D.) (c) Response time of [email protected] Probe toward NO (100 μM). (d) Selectivity and competition tests of [email protected] probe in the presence of NO (50 μM) and other ROS/RNS (100 μM) (n = 3, S.D.). Download figure Download PowerPoint Due to the complex environment of organisms in living cells, the selective and competitive tests of developed [email protected] probe were also investigated. No apparent effect (<4.6%) was observed from the common ROS, including H2O2, •OH, ClO−, O2•−, and 1O2 or RNS such as ONOO−, HNO, and NO2− (Figure 2d). Meanwhile, negligible influence (<4.2%) was found from amino acids and metal ions including K+ (100 mM), Na+ (50 mM), Ca2+ (10 mM), Mg2+, Fe3+, Fe2+, Cu2+, Cu+, and Zn2+ ( Supporting Information Figure S15). In addition, no obvious influence (<5.0%) was found for the competitive tests in the presence of NO and other potential interference of ROS/RNS, metal ions, or amino acids. The above tested results indicated the high selectivity of developed [email protected] toward NO, which benefited from the specific recognition part of RBD. Real-time imaging and quantification of NO in neurons Before applying the developed nanoprobe for biological sensing of NO, the cytotoxicity and biocompatibility of [email protected] nanoprobe were estimated. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) results proved that the cell viability was higher than 90% after the cells incubated with [email protected] nanoprobe (90 μg/mL) for 48 h ( Supporting Information Figure S16). In addition, flow cytometry assay results demonstrated that apoptotic cells (<6.2%) were seldom found after the cells incubated with the [email protected] probe (90 μg/mL) for 24 h ( Supporting Information Figure S17). The above results proved that the low cytotoxicity and high biocompatibility of the developed probe. Subsequently, the developed TPF probe [email protected] was further used for real-time biological imaging of NO in neurons. Colocalization imaging experiments proved that the [email protected] probe entered into neurons and primarily located in cytoplasm (Pearson’s correlation coefficient: 0.89) (Figure 3a). With the increasing concentrations of exogenous NO from 0 to 100 μM, the fluorescence of neurons from the red (Fred: 550–600 nm) gradually while that from the fluorescence (Fgreen: 430–500 nm) (Figure The intensity ratio of Fred to Fgreen (Fred/Fgreen) was increased from ± to ± (Figure It is that NO concentration in neurons was estimated to ± μM (n = S.D.) in the absence of exogenous NO based on the in which was with the previously reported Moreover, no apparent fluorescence increase was observed from the red under the stimulation exogenous NO (100 μM) in the presence of mM), a NO further the observed fluorescence increase in the red was ascribed to the concentration changes of NO. results proved that there was no between the results obtained from the NO probe and from our developed nanoprobe ( Supporting Information Figure and Table The above results showed that our developed [email protected] probe can be applied to biosensing and imaging of NO in neurons with high accuracy. Figure 3 | (a) Colocalization of neurons treated with [email protected] probe and The signal of probe was from to nm under the excitation of nm. The was from to nm at excitation of nm. The is the of the and the (b) TPF imaging of neurons incubated with [email protected] probe from different under the stimulation of different concentrations of NO (0, 50, and 100 and 100 μM NO with (c) fluorescence intensity ratio (Fred/Fgreen) obtained from (b) (n = S.D.). 25 Download figure Download PowerPoint Two-photon imaging of NO in neurons and brain under hypoxia is a typical of many including ischemia, and However, the mechanism of brain is To the in the brain, neuronal was first estimated under the stimulation of O2 concentration or of time resulted in neuronal death ( Supporting Information Figure The cell viability was to after hypoxia for 60 min. To the mechanism of hypoxia in neuronal the concentration of NO in neurons was further under hypoxia by using our developed [email protected] probe. With the of hypoxia time (Figure the fluorescence intensity of the red was while that of the indicating that hypoxia a increase in NO The concentration of NO increased to ± μM after hypoxia for min (Figure Moreover, flow cytometry assay results showed that after hypoxia for 15 min, of apoptotic neurons were while of apoptotic cells were found after hypoxia for min (Figure However, little apoptotic cells were found after neurons were stimulated by hypoxia for min in the presence of (Figure The results that NO plays an important as an in neuronal death by Figure 4 | (a) Two-photon imaging of neurons incubated with [email protected] probe after hypoxia for different (b) fluorescence intensity ratio (Fred/Fgreen) obtained from (a) (n = S.D.). 25 (c) The assay of neurons after hypoxia for different times (0, and hypoxia for min in the presence of and the regions of and neurons, (d) TPF imaging of NO in different brain regions and by using the developed [email protected] probe. fluorescence intensity ratio (Fred/Fgreen) obtained from (d) in different brain regions and S.D.). Download figure Download PowerPoint the other advantage of two-photon imaging with deep penetration, the concentrations of NO in different brain regions of brain under hypoxia were also estimated including of and As shown in Figure the concentrations of NO in brain regions were estimated to be ± However, after the brain were treated with hypoxia for min, the concentrations of NO in brain regions increased The concentration of NO in and increased to ± 0.3 μM and ± 0.3 μM, which was higher than that in ± μM) and ± μM). The results that different brain regions have different to results that our developed nanoprobe can be used for analysis of NO in neurons and brain In imaging of larval zebrafish under and To further the performance of the developed [email protected] probe for bioimaging in the 3-day-old larval zebrafish with was as living to high of with As shown in Figure TPF imaging of larval zebrafish displayed two fluorescence with a of much than the fluorescence ( Supporting Information Figure In of the that hypoxia resulted in the apparent decrease of the of zebrafish (Figure hypoxia induced an obvious increase of NO in the zebrafish brain (Figure and Supporting Information Figure The concentration of NO in the larval zebrafish brain under hypoxia was estimated to ± μM, which was higher than that in ± μM) (Figure The that zebrafish results further our developed [email protected] probe great potential in of bioimaging and biosensing of NO in Figure 5 | (a) two-photon confocal imaging of larval zebrafish incubated with [email protected] probe under the excitation of 800 nm. (b) of larval zebrafish after hypoxia for different (c) Two-photon confocal imaging of larval zebrafish brain incubated with [email protected] probe under and (d) fluorescence intensity ratio (Fred/Fgreen) obtained from (c) (n = 5, S.D.) Download figure Download PowerPoint A highly selective ratiometric TPF nanoprobe was developed for detection of the NO, with high stability and rapid response In of the of [email protected] nanoprobe, including high biocompatibility and low it was applied for real-time biosensing and bioimaging of NO in neurons, and it was demonstrated that hypoxia-induced neuronal death was regulated by NO. In addition, the that hypoxia-induced NO increase in various brain regions was different was also Moreover, we proved that death of larval zebrafish has a close relationship with the NO This a for understanding the mechanism of but also a to develop a highly stable metal fluorescent nanoprobe for ROS/RNS and other biological