Xin Huanga,
Zhipeng Lia,
Tingting Caoa,
Qian Caia,
Chengchu Zenga,
Hua Fub and
Liming Hu*ab
aCollege of Life Science and Bioengineering, Beijing Key Laboratory of Environmental and Oncology, Beijing University of Technology, Beijing, 100124, China. E-mail: huliming@bjut.edu.cn; Fax: +86 10 67392001; Tel: +86 10 67396211
bKey Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology (Ministry of Education), Tsinghua University, Beijing, 100084, China
First published on 18th April 2018
A methylene blue-based near-infrared fluorescent probe was designed for the selective determination of hypochlorite (ClO−), over other reactive oxygen species or interfering agents. Acetylated methylene blue was synthesized by introducing the acetyl group into the methylene blue framework, which can specifically recognize exogenous and endogenous ClO−. The acetylated methylene blue fluorescent probe was characterized by 1H NMR, 13C NMR and HRMS. The response process and possible mechanism were studied using products of the probe. The emission response of the probe to ClO− presented good linear relationship in the 0–60 μM concentration range, with the detection limit of 0.1 μM (measured at 660 nm and 690 nm). The absorption and emission wavelengths of acetylated methylene blue are both in the near-infrared region; in addition, the probe itself and the degradation products were well-dissolved in water and have almost no toxicity. The probe was used for intracellular ClO− imaging and showed a large fluorescence enhancement (about 200-fold increase).
In living organisms, ClO− is generated from hydrogen peroxide and chloride ion in activated neutrophils, catalyzed by myeloperoxidase (MPO).1,5,6 ClO− is commonly used in disinfectants and bleaches.7,8 However, excessive formation of hypochlorite can also cause tissue damage and a series of human diseases, such as atherosclerosis, arthritis and even cancers, etc.9,10 Therefore, the rapid and sensitive detection of ClO− is important in biological samples.
So far, many methods have been reported to detect ClO−, such as electrical analysis, potentiometric analysis, chemiluminescence, etc.11 Among them, the fluorescence method has the advantage of better sensitivity and selectivity.12–14 Fluorescent molecular probes can be employed as a powerful tool to track biomolecules in living systems due to their high sensitivity, real-time assay and non-invasive monitoring capability. In addition, the small-molecule fluorescent probe has many characteristics. The most attractive points are its low cost and low toxicity. Herein, a reactive fluorescent probe responsive toward ROS is designed.
Methylene blue (MB) is a photosensitizer approved by the FDA. This oxidized phenothiazine compound is widely used in clinical and basic research.15 Recently, MB was also applied as a NIR (near-infrared) imaging agent in image-guided surgery.16 As an ideal fluorescent group, the compound shows excellent photophysical properties, such as absorption and emission wavelength both in the NIR (more suitable for animal experiments to exclude background interference), almost no toxicity, and so on. So far, many new fluorescent probes have been reported based on near-infrared fluorophores.17–23
Here, we developed a highly sensitive and selective hypochlorite turn-on sensor (MBAC, Scheme 1). The probe was synthesized by acetylation of methylene blue and characterized by 1H NMR, 13C NMR and HRMS. Upon reaction with ClO−, the acetyl group leaves mbac, thus altering the fluorescent properties of the probe and achieving the detection of ClO−. Thus, MBAC is almost non-fluorescent; however, strong near-infrared fluorescence was detected from the ClO− response product. The advantage of mbac is that the emission wavelength is in the near-infrared region; especially, the fluorescence enhancement factor is large. The concentration is linearly related to the fluorescence intensity, and the detection limit is low.
We hypothesize that mbac amide bonds are cleaved by ClO−, resulting in the release of fluorescent group MB, while producing visible color changes and NIR emission (Scheme 1). This was verified by high-performance liquid chromatography (HPLC, Fig. 5). On the other hand, the experimental data show that MBAC was successfully utilized to detect endogenous hypochlorite in living cells, with excellent selectivity and sensitivity (the detection limit of 0.1 μM). According to its low cytotoxicity and high selectivity to ClO−, we see its application prospects and perform a series of tests on the probe.
At this stage, the water phase was khaki-colored, and the organic phase was dark blue. The organic phase was separated and dried over anhydrous sodium sulfate, followed by the addition of triethylamine (0.6 mmol, 85 μL) to form a mixture. 0.6 mmol of acetyl chloride was added to 1 mL of dichloromethane and stirred. Then, the triethylamine mixture was slowly added dropwise to prevent the production of a large amount of white smoke, followed by stirring at room temperature for 20 minutes. The synthesis of a compound with similar structure has been reported.24 The reaction process is shown in Scheme 2. The product was purified by column chromatography (alumina, dichloromethane).
Mbac (122 mg, 74.4%), white powder. 1H NMR (400 MHz, CDCl3) δ (ppm) 2.17 (s, 3H, CH3), 2.94 (s, 12H, CH3), 6.62 (s, 2H, Ar–H), 6.71 (d, J = 2.4 Hz, 2H, Ar–H), 7.26 (s, 1H, Ar–H), 7.42 (s, 1H, Ar–H). 13C NMR (100 MHz, CDCl3) δ 170.54, 169.28, 147.94, 109.62, 109.83, 39.61, 23.92. HRMS (ESI): m/z calcd for C18H21N3OS [M + H]+ 328.1484, found 328.1452.
The fluorescence intensity of the probe at different pH levels is shown in Fig. 2. It can be seen that the probe is relatively stable in the pH range of 3–10, and the most suitable pH for measurement is in the range of 5–8. Therefore, we chose the pH 7.4 for future experiments.
Fig. 2 Fluorescence intensity of the probe mbac (10 μM) with and without ClO− (500 μM) in water at different pH conditions, (λex = 660 nm). |
In addition, in order to investigate the stability of the probe, we measured the fluorescence intensity of the probe at different temperatures. The results show that the probe is relatively stable; the specific data are provided in ESI (Fig S5, Tables S1 and S2†).
In order to further explore the in vitro detection ability of mbac, the UV response of the probe to various concentrations of ClO− was studied. The UV-visible absorption spectra of the probe are shown in Fig. 4.
Fig. 4 UV of probe mbac (10 μM) upon the addition of ClO− (5–500 μM). The system was in PBS (pH = 7.4). |
As shown in Fig. 5, the fluorescent probe mbac has almost no emission in the visible region. After adding ClO− solution, the fluorescence intensity peak appeared near 690 nm and reached the maximum after incubation at room temperature for 15 min, stabilizing within 30 minutes. As shown in Fig. 4 and 5, the fluorescence intensity at 690 nm was significantly enhanced with the addition of ClO−. From almost colorless to blue, the process of change is visible to the naked eye (Scheme 1).
Fig. 5 Fluorescence intensity increase of probe mbac (10 μM) upon the addition of ClO− (5–500 μM), λex = 660 nm. The system was PBS (pH = 7.4). The fluorescence intensity was determined at 690 nm. |
The fluorescence excitation and emission wavelengths of mbac both reach the NIR range, which is very favorable for intracellular imaging. Fluorescence emission can be attributed to the cleavage of the amide bond to release the fluorescent group MB, which is confirmed by HPLC (Fig. S8†). The retention time of the probe was 12.87 minutes in HPLC, and for MB was 3.16 minutes. When ClO− was added to mbac, the peaks of mbac disappeared, while the peak of MB appeared.
In contrast, other ROS such as ˙OH, H2O2, t-BuOOH and NO˙; other ions such as K+, Na+, Li+, Hg+, Cs+, Ag+, Mg2+, Ca2+, Zn2+, Cu2+, Fe2+, Fe3+, Cl−, CH3COO−, Br−, NO3−, I−, H2PO4−, HPO42−, PO43−, NO2−, MnO4−, F−, SO42−, HCO3− and CO32−; and other reducing agents such as S2O42− and GSH showed very weak fluorescence enhancement (Fig. 6), even at 50 equivalents. All of the results demonstrate that the probe mbac exhibits high selectivity to ClO−.
The methyl thiazolyl tetrazolium (MTT) assay was used to measure the cytotoxicity of mbac in LO2 cells. LO2 cells were seeded into a 96-well cell-culture plate. Cells were dosed with mbac at final concentrations ranging from 6.25 μM to 100 μM in each well of the plates. The cell survival was determined by measuring the absorbance at 490 nm by using a microplate reader. A calibration curve was prepared using SPSS to determine the IC50 of probe-mbac. Cytotoxicity at IC50 is the concentration of compound at which the optical density of treated cells (48 h) is reduced by 50% with respect to untreated cells using the MTT assay. The cell viability at various mbac doses was 95.2%, 100.5%, 106.5%, 102.1% and 104.8%. The results are shown in Fig. S9;† mbac exhibits a relatively low cytotoxicity in LO2 cells after exposure to up to 100 μM mbac for 48 h.
On the other hand, a distinct emission response could be observed (Fig. 7b) after incubation with ClO− for 30 min. The results show that the cell permeability of the probe mbac is good. Thus, mbac can be used to estimate the concentration of intracellular ClO−.
MBAC was also expected to be cell-membrane permeable, per the calculated logP of 3.82 obtained with the ALOGPS 2.1 programme.25 The predicted value of logP is within the range indicating good capacity to cross the plasma membrane.26
Under physiological conditions, ClO− is highly reactive and short-lived; the average level of ClO− generation from neutrophils is 0.47 nmol min−1 per 106 cells.27 Thus, when only the probe is added, the fluorescence in the cell is almost invisible. Furthermore, we hope to use this probe to detect ClO− in living samples. The results are shown in Table 1; the fluorescence intensity of several samples is less than 10 a.u., tested in tap water andboiled water (Chaoyang, Beijing).
Samples | Average fluorescence intensity/a.u. | RSD |
---|---|---|
Tap water | 8.305 | 0.084889 |
Boiled water | 4.829 | 0.14549 |
ClO− | 348.705 | 0.000875 |
Blank | 2.185 | 0.009153 |
Furthermore, we found that boiled water contained less ClO− than tap water. The results show that mbac is a common tool that can be successfully applied to the detection of ClO− species in environmental samples.
Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c8ra01037d |
This journal is © The Royal Society of Chemistry 2018 |