Introduction

Ozone (O3), one of reactive oxygen species (ROS), has attracted much attention because of its key role in human health and disease1,2,3,4,5. Recently, O3 exposure has become a growing global health problem, especially in urban areas6,7. While O3 in the stratosphere protects the earth from harmful ultraviolet light, tropospheric or ground-level O3 is toxic and can damage the respiratory tract. Current evidence has demonstrated that ozone may be produced endogenously in inflammation and antibacterial responses of the immune system8,9,10. Moreover, cholesterol ozonolysis products are found to exist in clinical brain samples and likely trigger misfolding of protein in sporadic amyloid disease11,12. Thus, detection of O3 would be helpful to various biochemical studies as well as environmental evaluation. Toward this end, several fluorescent probes for O3 assay have been developed5,13,14, among which indigo carmine is a classical one, but it can react with a number of ROS, suffering from poor selectivity7. Moreover, some of these probes employ cyanine skeletons that are known to have poor stability and high background fluorescence due to ready autoxidation and photooxidation15; the others have a relatively short analytical wavelength or require organic solvents as cosolvents, which may disturb the normal function of biomolecules and are unfavorable for biological studies16. On the other hand, chemiluminescent probes are also proposed to measure O317,18,19. However, false-positive ozone readings are often reported because of poor selectivity of chemiluminescence18,19. Therefore, spectroscopic probes with superior properties, such as high selectivity and sensitivity and good stability and water solubility, are rather necessary for the detection of O3 but are still rare to the best of our knowledge.

In this work, we have developed such a probe (1; Fig. 1) that has the above desired properties by incorporating the but-3-enyl group specific for O3 into resorufin. We chose but-3-enyl as a recognition moiety because alkenes can be selectively cleaved by O3 via ozonolysis20,21,22,23 and resorufin as a fluorochrome due to its good stability and water-solubility as well as long analytical wavelength. Most notably, the fluorescence of resorufin is easily quenched via 7-hydroxy substitution24,25,26. Therefore, the designed 1, in which its 7-hydroxy is substituted by but-3-enyl, is anticipated to show no or weak color and fluorescence; upon reaction with O3, the resulting aldehyde (2) may undergo β-elimination20,21,22,23 to yield the fluorescent resorufin (Fig. 1). As a result, the fluorescence of the reaction system could be turned on, which may provide the basis for the sensitive detection of O3.

Figure 1
figure 1

Preparation of probe 1 and its reaction with O3.

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Results

Spectroscopic response of 1 to O3

The spectroscopic response of 1 to O3 was studied. As shown in Fig. 2A, probe 1 displays a moderate absorption peak at 484 nm with a shoulder at around 400 nm, but nearly no absorption at 574 nm. However, reaction of 1 with O3 gives a strong absorption band centered at 574 nm, concomitant with a distinct color change from nearly colorless to pink (see the inset of Fig. 2A), which may be useful for the simple detection of O3 by the naked eye. Fig. 2B depicts the fluorescence change of 1. As is seen, 1 itself shows a rather weak fluorescence around 585 nm, with a quantum yield of Φ ≈ 0.003 27, which is ascribed to the alkylation of the 7-hydroxy group of resorufin. This extremely low background signal is desirable for sensitive detection. However, addition of O3 leads to a 207-fold fluorescence enhancement, accompanied by a large fluorescence color change (see the inset of Fig. 2B). This fluorescence change is attributed to the release of the free resorufin (vide infra).

Figure 2
figure 2

Spectroscopic response of 1 to O3.

(A) Absorption spectra and (B) fluorescence emission spectra of 1 (10 μM) before (a) and after (b) reaction with O3 (30 μM). The absorption spectra were measured with 50 mM phosphate buffer (pH 7.4) as a blank. The insets show the images of color and fluorescence changes of 1 before and after reaction with O3.

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Optimization of experimental conditions

Reaction conditions (pH, buffer concentration and time) were examined in detail. The results showed that the most sensitive spectroscopic response of the probe to O3 can be achieved in the ranges of pH 7–10, 30–200 mM of the phosphate buffer concentration and 30–60 min of the reaction time (Supplementary Figs. S1–S3). As a result, a reaction medium of 50 mM phosphate buffer (pH 7.4) and a reaction time of 40 min at room temperature were chosen for the present system.

Analytical characteristics

Under the optimal conditions, 1 shows a gradual increase in the fluorescence intensity with the increase of O3 concentration (Fig. 3) and a good linearity between the relative fluorescence intensity and the O3 concentration was observed in the range of 1.0–30 μM O3, with a linear equation of ΔI = 66.3 × C (μM) + 22.6 (γ = 0.998). The detection limit (3 S/m, in which S is the standard deviation of blank measurements, n = 11 and m is the slope of the linear equation) was determined to be 5.9 nM O3, which corresponds to an ozone concentration of 0.056 mg m−3 in air in the present study (lower than the ambient air quality standard of 0.1 mg m−3 recommended by the World Health Organization). This detection limit is rather low, which is ascribed to the extremely low background signal of the probe.

Figure 3
figure 3

Fluorescence response of 1 to O3.

(A) Fluorescence response of 1 (10 μM) to O3 at varied concentrations (0, 1, 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 40, 45 and 50 μM); λex = 550 nm. (B) The plot of the relative fluorescence intensity (ΔI is the difference of fluorescence intensity of 1 in the presence and absence of O3) against the O3 concentration in the range of 1.0–30 μM. The measurements were performed in 50 mM phosphate buffer solution (pH 7.4) with λex/em = 550/585 nm.

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Selectivity studies

To assess the specificity of the reaction, the fluorescence response of 1 to various commonly coexisting substances in both environmental and biological systems (in particular in air and cells) were examined in parallel under the same conditions. The examined substances included ROS, air pollutants emitted from motor vehicles (e.g., lead, palladium, platinum, nitric acid and sulfuric acid that are derived from nitrogen dioxide and sulfur dioxide)28 and common cellular substances (e.g., CaCl2, MgCl2, glucose, vitamin C, amino acids, glutathione and proteins)29. As shown in Fig. 4, only O3 produces a large fluorescence enhancement, whereas the other substances do not show such a behavior. Moreover, the effects of these substances on the O3 assay were also investigated (Supplementary Table S1), revealing that these substances at considerable concentrations hardly interfere with O3 assay (error < 10%). These results indicate that 1 has a high selectivity for O3. The possible reason for this may result from the fact that only O3 can react with the terminal double bond of 1 to form a molozonide via a (3 + 2) cycloaddition20,21,22, which undergoes retro (3 + 2) cycloaddition to yield aldehyde 2, followed by β-elimination to release resorufin (Fig. 1).

Figure 4
figure 4

Fluorescence responses of 1 (10 μM) to various substances.

(A) ROS and air pollutants: blank (10 μM probe 1 only), H2O2 (100 μM), ClO− (50 μM), O2·−(10 μM), 1O2 (10 μM), ·OH (10 μM), HNO3 (100 μM), H2SO4 (100 μM), Pb2+ (100 nM), Pd2+ (100 nM), Pt2+ (100 nM), NaNO2 (250 μM), Na2SO3 (250 μM), Na2S2O3 (250 μM) and O3 (30 μM). (B) Common cellular substances: MgCl2 (100 μM), CaCl2 (100 μM), glucose (50 μM), vitamin C (10 μM), glutamine (50 μM), serine (50 μM), arginine (50 μM), glutathione (5 mM), bovine serum albumin (BSA, 100 μM), human serum albumin (HSA, 100 μM) and O3 (30 μM). λex/em = 550/585 nm. The results are the mean ± S.D. of three separate measurements.

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Reaction mechanism

To prove the fluorescence response mechanism, the reaction products of 1 with O3 were subjected to HPLC analysis. As shown in Supplementary Fig. S4, O3, resorufin and the probe have a retention time of 2.59 min (curve B), 7.37 min (curve C) and 17.07 min (curve D), respectively. Upon reaction with O3 (curve E), the peak of the probe at 17.07 min decreases largely, concomitant with the appearance of a major peak at 7.37 min characteristic of resorufin. This clearly indicates that the reaction of 1 with O3 generates free resorufin, thereby causing the generation of fluorescence (Fig. 1).

Detection of O3 in ambient air samples

Our goals also included that probe 1 could be utilized to develop a simple method for convenient detection of O3 in ambient air. To demonstrate this potential, we made the following experiments (Supplementary Fig. S5): the ambient air in a sealed box (40 cm × 30 cm × 30 cm) was irradiated by a UV-365 nm lamp for different periods of time (0–12 h) and then the air was sampled with a 50-mL syringe; after the air in the syringe was injected into a sealed test tube containing the phosphate buffer (pH 7.4) and probe 1, followed by reaction for 40 min, the fluorescence of the reaction solution was measured (Fig. 5A). Note that the full absorption of O3 from the O3-containing air in the phosphate buffer of pH 7.4 requires 10 min at least (Supplementary Fig. S6); in our work 15 min was used to absorb O3. For quantitative analysis of O3 at lower levels, an additional calibration curve was constructed in the concentration range of 0.1–0.5 μM O3 (Supplementary Fig. S7) and based on this curve the concentration of O3 in the air irradiated under UV-365 nm lamp for 3–12 h (Fig. 5A) was determined to be 0.16–0.39 μM, corresponding to 1.5–3.7 mg m−3 (Table 1).

Table 1 The concentration of O3 in the air irradiated by UV-365 nm lamp for different periods of time
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Figure 5
figure 5

Fluorescence intensity of 1 (10 μM) reacting with O3 from air.

(A) The air was irradiated by UV-365 nm lamp for different periods of time: 0 (control), 3, 6 and 12 h. (B) The air from four different places: (a) the northwestern 4th ring road of Beijing; (b) the garden of Institute of Chemistry, Chinese Academy of Sciences; (c) the outside of the laboratory window of Institute of Chemistry; (d) the inside of the laboratory window of Institute of Chemistry. Blank (another control) refers to the 50 mM phosphate buffer solution (pH 7.4) containing only probe 1. The measurements were made in 50 mM phosphate buffer solution of pH 7.4 with λex/em = 550/585 nm. The results are the mean ± S.D. of three separate measurements.

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Detection of O3 in ambient air from four different areas was further performed (see “Procedure for ozone assay in air” in Methods below). In brief, amber glass wide-mouth bottles containing water (typically 10 mL) were placed with the opening of the caps in different areas, naturally absorbing the ambient air for 8 h. The as-obtained samples were then subjected to fluorescence measurements according to the “General procedure for detection of O3” in Methods below. The obtained results are shown in Fig. 5B. As is seen, the four places have different O3 concentrations, among which the O3 concentration in the air from the northwestern 4th ring road is the highest, which may arise from the heavy traffic in this area.

Validation of the method

To evaluate the accuracy of the proposed method, a comparative study on the determination of O3 in both standard solutions and sample solutions prepared from the air of the northwestern 4th ring road of Beijing was made with iodometric titration. As shown in Table 2, the analytical results of standard solutions from the two methods are in good agreement with each other using a Student's t-test30, clearly indicating the validity of our probe. Furthermore, the content of O3 in the sample solutions (see Methods below for details) is not detectable by the iodometric titration due to its low sensitivity, but can be determined to be 0.21 μM with the present sensitive fluorescence method.

Table 2 Comparison of O3 concentration determined by the proposed fluorescence method and iodometric titration
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The recovery of O3 added into the sample solutions was examined. As shown in Supplementary Table S2, the recovery of O3 determined by the present method ranges from 97 to 110%. The above results indicate that 1 can be used as a sensitive and selective probe for O3 assay in real ambient air samples.

Application of the probe to cell imaging

First, the potential toxicity of 1 to cells was evaluated by using a standard MTT [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide] assay. The results (Supplementary Fig. S8) showed that the cell viability was not significantly changed upon treatment even with 10 μM 1 at 37°C for 24 h, indicating the low cytotoxicity and good biocompatibility of the probe. Then, the applicability of 1 for cell imaging was investigated. As shown in Fig. 6, the 1-loaded HeLa cells themselves exhibit neglectable intracellular background fluorescence (Fig. 6A), whereas the cells incubated with 5 μM O3 for 30 min display a strong red fluorescence (Fig. 6B). On the other hand, HeLa cells treated with 0.05 mM of ethyl 4-vinylbenzoate (a specific scavenger of O3)8 produce a decreased fluorescence (Fig. 6C) and a higher concentration of ethyl 4-vinylbenzoate (0.1 mM) leads to a weaker fluorescence (Fig. 6D). This suggests that the fluorescence change in HeLa cells indeed arises from the cleavage reaction of 1 by O3 releasing the free resorufin. Furthermore, to quantitatively compare the level of O3 in the presence and absence of ethyl 4-vinylbenzoate, relative pixel intensity analysis was made by using ImageJ software (version 1.45 s, NIH), which revealed that the fluorescence intensity from the cells treated with 0.05 and 0.1 mM of ethyl 4-vinylbenzoate was decreased to 35% and 17%, respectively, compared with that (defined as 100%) in the absence of ethyl 4-vinylbenzoate (Supplementary Fig. S9). The above results indicate that the probe is cell membrane permeable and can also be used to detect the change of O3 in cells.

Figure 6
figure 6

Confocal fluorescence images of HeLa cells.

(A) HeLa cells were incubated with 10 μM 1 for 30 min (control); (B) the 1-loaded HeLa cells were incubated with 5 μM O3 for 30 min; (C) the 1-loaded HeLa cells were treated with 0.05 mM ethyl 4-vinylbenzoate for 30 min and then incubated with 5 μM O3 for 30 min; (D) the 1-loaded HeLa cells were treated with 0.1 mM ethyl 4-vinylbenzoate for 30 min and then incubated with 5 μM O3 for 30 min. The differential interference contrast (DIC) images of the corresponding samples are shown below (images E-H). Scale bar, 10 μm.

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Discussion

As mentioned above, exposure to environmental O3 is particularly a problem for those who suffer from respiratory ailments. These patients rely solely on community-based monitoring to adjust their indoor or outdoor activity so as to minimize their exposure. Because of this, development of a simple and sensitive method for O3 assay is rather necessary for not only various biochemical studies but also air quality forecast.

By taking advantage of both the excellent fluorochrome of resorufin and the specific reaction of but-3-enyl with O3, we have established such a simple and practical method with a detection limit of 0.056 mg m−3 in air (lower than the ambient air quality standard of 0.1 mg m−3 recommended by the World Health Organization) through designing a resorufin-based spectroscopic off-on probe. The probe shows an extremely low background spectroscopic signal, but reacts with O3 producing a distinct color and fluorescence change, which makes the probe suitable for sensitive assay of O3. This applicability has been successfully demonstrated on both the determination of O3 in real ambient air samples and the imaging of O3 in cells. We believe that the probe is worth popularizing and may further find important analytical applications in practice.

Methods

Synthesis of 1

To a suspension of resorufin sodium salt (0.24 g, 1.0 mmol) in anhydrous DMF (5 mL), K2CO3 (0.21 g, 1.5 mmol) was added. After stirring for 30 min under Ar atmosphere, a solution of 4-bromo-1-butene (0.27 g, 2.0 mmol) in DMF (2 mL) was added dropwise. The resulting mixture was stirred at 40°C for 2 h and then diluted with dichloromethane (50 mL). The reaction solution was washed three times with water (50 mL × 3) and brine (50 mL × 3) successively. The organic layer was separated and dried over MgSO4. The solvent was removed by evaporation and the residue was subjected to silica gel chromatography eluted with petroleum ether (b.p. 60–90°C)/ethyl acetate (v/v, 1:1), affording probe 1 as an orange solid (0.14 g, 58%). 1H NMR (300 MHz, CDCl3; Supplementary Fig. S10): δ 7.70 (d, J = 9.0 Hz, 1 H), 7.42 (d, J = 9.9 Hz, 1 H), 6.92–6.96 (m, 1 H), 6.81–6.86 (m, 2 H), 6.33 (d, J = 2.1 Hz, 1 H), 5.84–5.97 (m, 1 H), 5.14–5.24 (m, 2 H), 4.10–4.15 (m, 2 H), 2.57–2.64 (m, 2 H); 13C NMR (75 MHz, CDCl3; Supplementary Fig. S11): δ 186.3, 163.0, 149.8, 145.7, 145.5, 134.7, 134.2, 133.5, 131.6, 128.4, 117.7, 114.0, 106.7, 100.6, 68.2, 33.3. EI-MS (m/z): [M]+ calcd. for C16H13NO3, 267.09; found, 267 (Supplementary Fig. S12). Analysis (calcd., found for C16H13NO3): C (71.90, 71.52), H (4.90, 5.08), N (5.24, 5.10).

General procedure for detection of O3

Unless otherwise noted, all the measurements were made according to the following procedure. In a 10 mL test tube, 5 mL of 50 mM phosphate buffer (pH 7.4) and 100 μL of the stock solution of 1 were mixed, followed by addition of an appropriate volume of O3 solution. The final volume was adjusted to 10 mL with the phosphate buffer and the reaction solution was mixed well. After 40 min at room temperature, a 3-mL portion of the reaction solution was transferred to a quartz cell of 1 cm optical length to measure absorbance and fluorescence intensity/spectrum with λex/em = 550/585 nm and both excitation and emission slit widths of 10 nm. In the meantime, a blank solution containing no O3 was prepared and measured under the same conditions for comparison.

Procedure for ozone assay in air

Amber glass wide-mouth bottles containing water (typically 10 mL) were placed with the opening of the caps in four different areas [(a) the northwestern 4th ring road of Beijing; (b) the garden of Institute of Chemistry, Chinese Academy of Sciences; (c) the outside of the laboratory window of Institute of Chemistry; (d) the inside of the laboratory window of Institute of Chemistry], naturally absorbing the air of the corresponding area for 8 h. Care was taken to ensure that the samples were not hit by direct sunlight. The as-obtained samples were then subjected to fluorescence measurements according to the above “General procedure for detection of O3”. For each place, experiments were done in triplicate.

Determination of ozone by iodometric titration

The determination of ozone by iodometric titration was carried out according to the known method31. Briefly, in a 100 mL flask, 20 mL of KI solution (100 μM) was mixed with 20 mL of the phosphate buffer (pH 6.0) containing O3, followed by stirring for 30 min. Then, diluted H2SO4 was added dropwise to adjust the pH to 2.0. After 5 min, the solution was titrated with Na2S2O3 solution (10 μM). When the color of the reaction solution became light yellow (near the end point), a few drops of starch solution were added and the titration continued until the violet blue color disappeared due to the change of iodine to iodide.

Fluorescence imaging of ozone in HeLa cells

HeLa cells were grown on glass-bottom culture dishes (MatTek Co.) using Dulbecco's modified eagle media (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS) and 100 μg mL−1 penicillin-streptomycin in a humidified 37°C, 5% CO2 incubator. Before use, the adherent cells were washed three times with FBS-free DMEM. For O3 imaging, the cells were incubated with 10 μM of 1 in DMEM at 37°C for 30 min, then with 5 μM of O3 for 30 min and finally washed three times with PBS (pH 7.4). Further researches were made by pretreatment of cells with ethyl 4-vinylbenzoate, a specific scavenger of O3. Briefly, the cells were first incubated with 10 μM of 1 in DMEM at 37°C for 30 min, washed with PBS and then incubated with ethyl 4-vinylbenzoate at different concentrations (0.05–0.1 mM) for 30 min. After that, the pretreated cells were incubated with 5 μM of O3 for 30 min and then washed three times with PBS (pH 7.4) for fluorescence imaging.