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Endoplasmic reticulum directed ratiometric fluorescent probe for quantitively detection of basal H2O2 Congcong Gao, Yong Tian, Ru-Bo Zhang, Jing Jing, and Xiaoling Zhang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b03809 • Publication Date (Web): 13 Nov 2017 Downloaded from http://pubs.acs.org on November 13, 2017
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Endoplasmic reticulum directed ratiometric fluorescent probe for quantitively detection of basal H2O2 Congcong Gao, Yong Tian, Rubo Zhang*, Jing Jing*, Xiaoling Zhang* Key Laboratory of Cluster Science of Ministry of Education, Beijing Key Laboratory of Photoelectronic/Electrophotonic Conversion Materials, School of Chemistry, Beijing Institute of Technology, Beijing 100081, PR China. Corresponding author. Tel/fax: +86 010 88875298. E-mail addresses:
[email protected],
[email protected],
[email protected]. ABSTRACT: The ER has a central role in the fine tuning of environmental and internal stimuli. We herein report a ratiometric fluorescent probe, α-Naph, capable of determining basal H2O2 in ER. The probe specifically response to H2O2. The LOD of the probe is as low as 38 nM, making it a feasible sensor to image intracellular basal H2O2. In addition, utilizing its ratiometric property, we are able to measure the concentration of H2O2 in ER quantitatively, eliminating the error caused by probe concentration and environment. The intracellular concentration of H2O2 in ER is calculated to be 0.692 µM under normal conditions and 1.26 µM under the stimulation stimulation of PMA.
Hydrogen peroxide(H2O2) is one of the most critical reactive oxygen species, which is involved in a wide range of physiological and pathological processes in human health and aging, including cell proliferation, differentiation, and migration.1 Emerging evidences reveal that the various roles of H2O2 acting in protein folding, signaling, respiration, metabolism or defense response greatly depends on its location within cells. As thus, instead of evaluating the H2O2 at a whole single cell level, monitoring the subcellular distribution and trafficking of H2O2 offers us an opportunity to decipher relationships between its complicated cellular chemistry and downstream biological effects. Fluorescent probes are now commonly used due to their noninvasive biological molecules imaging and processes with high spatial and temporal resolution. 2-4 To better understand the contributions of subcellular localization and trafficking of H2O2 in physiological
and pathological processes, probes that can target to specific subcellular spaces have been vigorously developed, including mitochondria,5-12 lysosome,13-16 and the nucleus.17, 18 The endoplasmic reticulum (ER) has been regarded as an oxidizing environment for the central role in the synthesis and distribution of proteins. In ER, proteins always form a proper folding in high redox state with disulphide bonds before being transported to their destination. ER homeostasis can be perturbed by an accumulation of misfolded and unfolded proteins in the ER lumen, a condition termed as ER stress. When misregulated, the accumulation of H2O2 may cause oxidative damage to cellular proteins. It can even trigger apoptosis by cleavage of caspase-12 in ER,19-21 resulting in diseases such as neurodegenerative and cardiovascular diseases, metabolic diseases, and cancer.22, 23 Thus, developing effec1
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tive analytical method for H2O2 in ER is of great importance. In 2010, Christopher J. Chang group reported a boronate-capped dye to visualize exogenous H2O2 in ER based on SNAP-AGT bioconjugation chemistry. However, quantitative detection of endogenous H2O2 in ER remains challenge. To address this issue, we synthesized a simple but effective probe (α-Naph), based on the chemical properties of α-ketoamide24, 25 in combination with naphthalimide, utilizing ratiometric detection scheme for the sensitive determination of basal H2O2 in ER. Hydrogen peroxide react with α-ketoamide in alkaline organic solvents, and followed by hydrolysis of amido linkage. 25 Using ratiometric approaches, we are even capable calculating more accurate and quantitative concentrations of H2O2 by simultaneously monitoring two signals. The good selectivity, ultra-sensitivity as well as specific ER targeting ability of α-Naph towards H2O2 makes it a perfect candidate to visualize basal H2O2 in ER in living cells. The rational design of fluorescent probe candidate for H2O2 was shown in Scheme 1.
Scheme 1. Design strategy for endoplasmic reticulum-specific hydrogen peroxide reporter based on naphthalimide. EXPERIMENTAL SECTION Materials and Instruments The 4-amino-1, 8-naphthalimide was prepared according to a reported procedure.27 All the chemical and biological reagents were obtained from commercialized companies and prepared in
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stock with standard methods before use. Silica gel (200-300 mesh, Qingdao Haiyang Chemical Co.) was used for column chromatography. 1H and 13C NMR spectra were recorded on a Bruker Avance III at 400 MHz or at 100 MHz. δ values are in ppm relative to TMS. Mass spectra (MS) were measured with Bruker Apex IV FTMS using electrospray ionization (ESI). Absorption spectra were recorded on a Purkinje TU-1901 spectrophotometer. Fluorescence measurements were taken on a Hitachi F-7000 fluorescence spectrometer with a 10 mm quartz cuvette. pH measurements were carried out with a pH acidometer (Mettler Toledo FE-30). Synthetic methods Synthesis of Compound 2 Compound 2 was prepared by adapting published procedures.28 SnCl2·2H2O (2.800 g, 3 mmol) in concentrated hydrochloric acid (15 mL) was added dropwise to a stirred cloudy solution of compound 1 (0.500 g, 0.5 mmol) in ethanol (25 mL) at room temperature. The reaction was heated to reflux for 8 h. After cooling down to room temperature, aqueous solution of Na2CO3 (10%) was added to quench the reaction. The precipitate was collected by filtration, washed with water (3 × 50 mL), and dried in vacuo to afford the crude product 2, which was directly used for the preparation of compound 3 without further purification. Synthesis of Compound 3 N-butylamine (0.5 mL, 1 mmol) was quickly added to a cloudy solution of 2 (0.500 g, 0.5 mmol) in ethanol (30 mL). After refluxing for 8 h, the reaction was cooled to room temperature. The solvent was removed under reduced pressure, and the crude product was purified by silica gel column chromatography (dichloromethane / acetone: 5/1) to give a brown yellow solid of compound 3. Synthesis of α-Naph α-Naph was synthesized according to a reported procedure.21 A mixture of 2-(4-nitrophenyl)-2oxoacetic acid (0.140 g, 0.2 mmol), oxalyl chloride (186 µL, 0.6 mmol), DMF (3 drops), and di2
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chloromethane (5 mL) was refluxed for 1 h and then evaporated. To this crude product, 5 mL of dichloromethane was added, and then Compound 3 (0.100 g, 0.1 mmol) and triethylamine (200 µL, 0.4 mmol) were added. The reaction mixture was stirred at room temperature for 30 min, poured into saturated NaHCO3 solution (50 mL), and extracted with 50 mL dichloromethane three times. The extracts were combined and the organic solvent was dried over anhydrous sodium sulfate and evaporated under reduced pressure. The residue was purified by column chromatography on silica gel (dichloromethane/ Methanol, 100:1 v/v) to afford a yellow solid (0.200 g, 83% yield). 1H NMR (400 MHz, DMSO-d6) δ = 11.53 (s, 1H), 8.63 (d, 1H), 8.61 – 8.51 (m, 2H), 8.49 – 8.41 (m, 2H), 8.40 – 8.32 (m, 2H), 8.31 – 8.25 (m, 1H), 7.97 – 7.90 (m, 1H), 4.11 – 4.03 (m, 2H), 1.69 – 1.56 (m, 2H), 1.43 – 1.29 (m, 2H), 1.06 (t, 3H). 13C NMR (126 MHz, DMSO-d6) δ 187.57 , 163.84 , 163.45 , 163.35 , 151.05 , 138.83 , 137.96 , 132.05 , 131.57, 129.88 , 128.72 , 127.48 , 125.73 , 124.44 , 122.87 , 122.29 , 119.99 , 109.98 , 55.37 , 30.13 , 20.28 , 14.21. HRMS (ESI) m/z: [M+H]+ calcd for 446.1274; found 446.1345. General procedures for analysis A parent stock solution of probe α-Naph (1.0 mM) was prepared in dimethyl sulfoxide (DMSO). The working standards were prepared by placing 10 µL of the parent stock solution and the appropriate volume of other solution into a test tube, then diluting the solution to 1 mL the DMSO: PBS (3:7, v/v, PBS, 20 mM, pH = 7.4). All spectra were obtained in a quartz cuvette (path length = 1 cm). Determination of the detection limit Referring to our previous paper, the detection limit was calculated based on the fluorescence titration,29 carried out in DMSO: PBS (3:7, v/v), pH = 7.4, using the following Eq. (1):
Detection limit =3σ / k
(1) Where σ is the standard deviation of blank measurements, and k is the slope of the plot of fluorescence intensity vs H2O2 concentration.
Endogenous H2O2 imaging in live cells with αNaph HeLa cells were seeded in high-glucose DMEM supplemented with 10% fetal bovine serum, 1% penicillin, and 1% streptomycin. Cultures were maintained in a humidified incubator at 37 °C, in 5% CO2/95% air. Fluorescence imaging of HeLa cells was observed under OLYMPUS FV1000 confocal fluorescence microscope with excitation wavelength fixed at 405 nm and fluorescence wavelengths at 420—510 nm (blue channel) and 510—610 nm (green channel). In addition, combined with Fig. 2B, we roughly calculated the endogenous H2O2 content in HeLa cells. For colocalization assay, cells were treated and incubated with 5 µM ER-tracker Red and 5 µM α-Naph at 37 °C under 5 % CO2 for 30 min. Excitation wavelength of ER-tracker Red was at 543 nm and fluorescence wavelengths at 620—700 nm. Results and discussion The influence of pH on the properties of αNaph was first examined in the absence and presence of H2O2. The nucleophilic addition was sensitive to pH, see Fig. S1. The hydrogen peroxide anion, HOO–, is much more nucleophilic in alkaline conditions.29 Reactions with peroxide are further facilitated by the fact that a greater fraction exists in the reactive, anionic form at physiological pH (H2O2, pKa = 11.6).29 The spectral response of α-Naph to H2O2 was evaluated in PBS buffer (20 mM, pH =7.4, containing 30% DMSO, v/v). In the absence of H2O2, α-Naph displayed one major absorption band centered at 365 nm with a corresponding blue-colored fluorescence maximum at 465 nm. After adding 100 µM H2O2, a new absorption peak appeared at 458 nm (Fig. 1A). The probe displays extremely weak fluorescence and generate strong green fluorescence signals due to the generation of 4-amino-naphthalimide (fluorophore of α-Naph) when reacting with H2O2 (Fig. 1B). The changes of fluorescence spectra is a result of the cleavage of α-ketoamide moiety and the simultaneous production of stronger electron3
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donating amino cation, as shown in Scheme 1. To further confirm the reaction process of α-Naph with H2O2, the purified reaction product was characterized to be 4-amino naphthalimide via ESI-HRMS (m/z 269.1185 for [M + H]+) (see Scheme S1 and Fig. S9). The proposed reaction mechanism for α-Naph and H2O2 was shown in Scheme S2.
Figure 1. (A) Absorption spectra of α-Naph (100 µM) in the absence and presence of H2O2 (100 µM) (B) Fluorescence spectra of α-Naph (10 µM) in the absence and presence of H2O2 (10 µM). The measurements were performed in DMSO: PBS= 3:7 (v/v), pH = 7.4, at room temperature. Excitation wavelength = 395 nm. Quantification of H2O2 and detection limit The reaction kinetics was investigated through fluorescence spectral analysis. Upon gradually increasing the concentration of H2O2, the fluorescence band centered at around 540 nm increased and the band centered at 465 nm decreased (Fig. 2A). The relative fluorescence emission intensity (540/465 nm) at various H2O2 concentrations (0−1 µM) was determined and shown in Fig. 2B. Fluorescence spectra of H2O2 concentration varies from 0−10 µM was shown in Fig. S2). The limit of detection (LOD) value was calculated to be 37 nM based on S/N = 3, which hinted that αNaph was sensitive enough to image endogenous H2O2 in bio-samples.
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µM); (B) The integrated fluorescence intensities ratio of α-Naph (10 µM), ranges from 510-610 nm to 420-510 nm. Abscissa are the concentration of H2O2.The measurements were performed in DMSO: PBS= 3:7 (v/v), pH = 7.4, at room temperature. The selectivity of α-Naph The specificity of α-Naph toward H2O2 was evaluated. The probe was incubated with various reactive oxygen species (ROS), reactive nitrogen species (RNS), and reactive sulfur species (RSS) including 1O2, ONOO−, ClO−, NO, tert-butyl hydroperoxide (TBHP), •OH, O2•−, cysteine (Cys), glutathione (GSH), hydrogen sulfide (H2S), and homocysteine (Hcy) which may interfere in sulfonic ester or boronate based probes.30-36 As can be seen, only H2O2 induces a dramatic fluorescence enhancement, while other ROS, RNS, and RSS trigger limited changes (Fig. 3). Additionally, other biological relevant species including cations (Na+, K+, Ca2+, Zn2+, Cu2+, Co2+, Mg2+, Fe2+, and Fe3+), anions (NO3−, NO2−, CH3COO−, S2O32−, PO43−, CO32−, SO32−, and SO42−) do not cause any changes of the fluorescence intensity (Fig. S3). These results demonstrated that α-Naph can detect H2O2 with high specificity. Fluorescence Imaging of ER H2O2 in HeLa Cells To confirm the specific stain of α-Naph, a colocalization experiment was performed. HeLa cells were costained with α-Naph and ER-Tracker Red, a commercially available endoplasmic reticulum marker. As displayed in Fig. 4, the images of red channel and green channel merged well. Moreover, the Pearson’s colocalization coefficient was calculated to be 0.9, indicating that αNaph was organelle-specifically trapped in the ER.
Figure 2. (A) Fluorescence response of α-Naph (10 µM) to varied concentrations of H2O2 (0-1 4
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Figure 3. Fluorescence responses of the probe (10 µM) toward various ROS/RNS/RSS: (100 µM): (1) Cys; (2) GSH; (3)Hcy; (4) H2S; (5) O2•− ; (6) ClO−; (7) TBHP; (8)ONOO −; (9) •OH; (10) 1 O2; (11) NO; (12) H2O2 (10 µM).
Figure 4. HeLa cells were incubated with ERTracker Red (100 nM, 10 min) (a) and α-Naph (5 µM, 10 min) (b and c are blue and green channel, respectively). (d) Merged image. Scale bar: 20 µm. Next, we assessed whether α-Naph could be used to detect endogenous H2O2 in the ER of living cells under confocal laser fluorescence microscopy. To this end, we treated HeLa cells with 5 µM of α-Naph for 5 minutes. Confocal laser fluorescence images for α-Naph (green：510— 610 nm and blue: 420—510 nm) are shown in Fig. 5A. When H2O2 scavenger N-acetylcysteine (NAC) was initially incubated with HeLa cells for 30 minutes, the fluorescence in blue channel increased and the fluorescence in green channel decreased, as shown in Fig. 5B. As shown in Fig. S5, there is no fluorescence in HeLa cells without any treatment (control), and then a strong green signal come into view because of the fluorescence enhancement induced by basal H2O2 (Fig. 5A). Meanwhile, as expected, the fluorescence was distinctly attenuated in the presence of NAC, which was widely used to eliminate H2O2 in living cells (Fig. 5B) .37 To further prove the reactivity of α-Naph with H2O2,
after incubated with NAC, HeLa cells were stimulated by phorbol myristate acetate (PMA), a H2O2 inducer through a cellular inflammation response.38, 39As can be seen from Fig. 5C, a strong green fluorescence was recovered. The ratio image of the HeLa cells labeled with αNaph constructed from two collection windows gave an average emission ratio of 1.34 (Fig. 5F and 5G-A). More importantly, α-Naph was responsive to the changes in the H2O2 concentration: the Fgreen/Fblue ratio increased to 2.24 when the cells were preincubated for 1 hour with PMA (Fig. 5G-C), which increases H2O2 production. The ratio also decreased to 0.47 upon treatment with NAC (Fig. 5G-B). Variations of the relative intensity of α-Naph were the manifestation of the change with intracellular H2O2, which hinted that the probe can be a sensor to endogenous H2O2.
Figure 5. Fluorescence images of the basal H2O2 in HeLa cells (a, b and c are blue channel, green channel and merged images of A, B and C 5
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with their bright field, respectively). (A) Cells stained with the probe (5 µM, 5 min). (B) Cells pretreated with NAC (10 mM, 1 h) and then stained with the probe (5 µM, 5 min). (C) Cells pretreated with NAC (10 mM, 1 h) and then PMA (1 mg/mL, 1 h), after that stained with the probe (5 µM, 5min). (D) Intensity analysis along the line in selected HeLa cells in A. (E) Relative intensity of ROI in (D). Scale bar: 20 µm. Intensity analysis and Relative intensity of ROI in (B) and (C) see Fig. S6 and S7. (F) The ratio images generated by Olympus software, and the color strip on the right represents the pseudocolor changes with α-Naph (A). (G) Relative fluorescence intensities of A, B and C. And changes of endogenous H2O2 stimulated by PMA were displayed in Figure 6A. HeLa cells were eliminated H2O2 by NAC, and then collected the fluorescence signal when PMA was added. With the time of stimulation increasing, the concentration of H2O2 showed a certain regular tendency of rising (Fig. 6B). These results suggested that α-Naph was capable of detecting endogenously stimulated H2O2 in the cells. Having demonstrated the satisfactory property for basal H2O2, α-Naph was then used to detect and figure out the ER concentration of H2O2. According to Fig. 5G-A, the Fgreen/Fblue ratio is 1.34, substituting it into the formula F510− 610 / F420−510 = 1.58364 X + 0.24273 ( Fig. 2B), the content of basal H2O2 is 0.692 µM, which is consistent with the literature reports that H2O2 is generated at a low concentration (
