A BODIPY-Based Far-Red-Absorbing Fluorescent Probe for Hypochlorous Acid Imaging

Abstract

Hypochlorous acid (HClO) is produced by white blood cells to defend against injury and bacteria. However, as one of the reactive oxygen species, high intracellular HClO concentration could lead to chronic diseases that affect the cardiovascular and nervous systems. To monitor HClO concentrations in bio-samples, the fluorescent probe is preferred to have: a) absorbability in the far-red window with reduced light-toxicity and improved tissue penetration depth, b) ratiometric feature for accurate analysis. In this study, we reported a far-red ratiometric HClO fluorescence probe based on BODIPY chromophore and aldoxime sensing group. Not only the color change of the probe solution can be detected by naked eyes, but also the emission ratios (I₆₄₅/I₆₇₀) showed a significant increase upon the introduction of HClO. More importantly, the feasibility of HClO monitoring in bio-samples was demonstrated in vitro using a confocal microscope.

Graphical Abstract

1. Introduction

Hypochlorous acid (HClO) is a compound frequently encountered within the mammalian immune system. Hypochlorous acid is often produced in the myeloperoxidase-catalyzed (MPO) reaction of hydrogen peroxide and chloride ions within phagocytes and neutrophils [1]. Despite its involvement in the functioning of phagocytes, neutrophils, and other antitumor cellular processes, high intracellular HClO levels have been linked to chronic diseases that affect the cardiovascular and central nervous systems in mammals. Thus, developing an efficient tool to monitor intracellular HClO concentration benefit the research involving the prevention and treatment of chronic diseases caused by increased HClO such as atherosclerosis [2] and lung injury [3]. The conventional method of HClO detection utilizes sodium thiosulfate and potassium iodide-starch indicators, cannot be applied to living cells, and cannot detect micromolar levels of HClO [4].

Fluorescent dyes have been widely used in chemosensors [5], photodynamic therapy [6], 3D data storage [7], and biological imaging [8] due to their advantages in spatiotemporal control and selective response. Fluorescent probes with the real-time response of HClO have also been developed [9]. Typically, these probes consist of two components: an HClO-sensitive functional group and a chromophore group capable of fluorescence in response to changes in the functional group. The selection of HClO-sensitive functional groups include p-methoxyphenol [10], hydroxylamine [11] and chalcogenide [12]. Among them, the fluorescent probes with aldoxime group conjugated with BODIPY [13], rhodamine [14], and phenanthroline [15] have exhibited great response. In particular, the aldoxime group can react with HClO to generate aldehyde, carboxylic acid, or nitrile oxide, which leads to the structure convert oriented emission change.

Though much progress has been made, the development of ratiometric far-red fluorescence probes for bio-imaging has been limited [16]. Fluorescent dyes with an absorbance maximum approaching far red-to-near infrared (NIR) range have adequate ability to perform sensing in deep body tissues [17]. Among the many far-red chromophores, boron-dipyrromethene (BODIPY) derivatives is an important family, as it has superior photo-stability, high fluorescent quantum yield, relatively high stability, and high absorption extinction coefficient [18]. In addition, the synthesis and characterization of the BODIPY structures have been well developed in past decades [19]. Therefore, various BODIPY derivatives are developed as photodynamic therapy agents, photocatalysts, and optical sensors. For instance, BODIPY-based probes have been developed for detecting metal ions [20, 21], pH [22], reactive oxygen species[23], and completing drug release upon light irradiation [24]. Furthermore, to quantify the concentration of HClO in biosamples, a good ratiometric property is demanded for accurate measurement. For example, Yin et al. reported a ratiometric fluorescent probe utilizing BODIPY and pyrrole group to achieve the emission response wavelength near 580 nm and demonstrated its high fluorescence ratio enhancement with the presence of HClO [25].

Herein, we conjugated aldoxime with a far-red absorbable BODIPY chromophore that has an extended conjugation system. The two-component system was designed to include a novel far-red fluorescent probe and an HClO-sensitive functional group to achieve selectivity towards HClO in the biological friendly excitation window (Scheme 1). Importantly, this oxime probe (BD-NOH) showed a ratiometric property with a ratio of emission intensity (645 nm vs. 670 nm) that experienced a significant enhancement in the presence of HClO. Overall, this is the first report of a far-red BODIPY probe that can detect intracellular HClO concentration in a ratiometric manner.

Scheme 1.

Synthesis route of HClO probe (BD-NOH). The labels correspond to the following: a, DMF, piperidine, acetic acid, microwave irradiation 150 °C, 40 psi, 5 min; b, POCl₃, DMF, dichloromethane; c, NH₂OH HCl, ethanol.

2. Materials and methods

2.1. Materials

5% Sodium hypochlorite (NaClO) commercial aqueous solution was used to prepare the diluted solutions. All the solutions were prepared by reagents with analytical grades and distilled water. Direct dissolution of appropriate amounts of SO₄²⁻, Cl⁻, NO₃⁻, ClO₄⁻, F⁻ sodium salts were used to prepare stock solutions (0.5 mmol/L). All chemicals were purchased from Fisher Scientific^®. Solvents were used directly from purchase from Fisher Scientific^® and Sigma Aldrich^®.

2.2. Instruments

The pH is measured with a pH meter (Fisher Scientific accumet^® AB150). TLC analysis was completed with pre-coated silica gel plates. A Bruker AVANCE spectrometer (500 MHz) was used to acquire ¹H and ¹³C NMR spectra in CDCl₃ solution. Orbitrap mass spectrometry analysis was conducted in the Prof. Hao Chen lab, Department of Chemistry and Environmental Science, New Jersey Institute of Technology (NJIT). An Agilent 8453 spectrophotometer was used to acquire UV-vis absorption spectra. An FLS980 fluorescence spectrometer was used to record fluorescence emission spectra. Cell images were recorded using a Zeiss LSM 780 confocal microscope. Fiji, a freely available image processing software, was used to process all the cell images.

2.3. Preparation and characterization of probes

2.3.1. Compound 1

Trimethoxy-BODIPY (50 mg, 0.12 mmol) and diphenyl sulfide aldehyde (50 mg, 0.23 mmol), which were both synthesized by the formerly reported method [26], were added to a 10 mL glass vessel along with DMF (2 mL), acetic acid (0.2 mL) and piperidine (0.2 mL). The vessel was inserted into a CEM microwave reactor and reacted at a temperature of 150 °C and pressure of 40 psi for 5 min. The solution was then washed with brine solution and the organic solvent was removed via vacuum pump. The mixture of products was purified by column chromatography with an eluent consisting of hexane/ethyl acetate (4:1, v/v) to afford compound 1 as purple solid (40 mg, 41% yield). ¹H NMR (500 MHz, CDCl₃) δ: 7.78 (d, 2H), 7.54 (d, 4H), 7.42 (d, 4H), 7.37–7.28 (m, 10H), 7.20 (d, 2H), 6.65 (s, 1H), 6.57 (s, 1H), 3.93 (s, 3H), 3.85 (s, 6H), 1.60 (s, 6H). ¹³C NMR (126 MHz, CDCl₃) δ: 154.17, 152.54, 142.11, 138.74, 138.52, 137.77, 135.36, 135.04, 134.65, 133.44, 132.01, 130.19, 129.40, 128.14, 127.68, 119.30, 117.87, 105.58, 61.40, 56.44, 29.73, 14.51.

2.3.2. Compound 2

1 mL POCl₃ and 1 mL DMF was added to round bottle flask in ice bath and stirred for 5 min, then 50 mg of Compound 1 in DCM solution was added and the mixture was refluxed for 1 h. The reaction mixture was washed with a brine solution and after extraction the organic solvent was removed via vacuum pump. Then product mixture was purified by column chromatography with ethyl acetate/hexane (1:3, v/v) as the eluent to afford a purple solid of 45 mg (87% yield). ¹H NMR (500 MHz, CDCl₃) δ: 10.11 (s, 1H), 7.69 (t, 2H), 7.54 (t, 3H), 7.47–7.42 (q, 4H), 7.38–7.31 (m, 10H), 7.18 (d, 2H), 6.78 (s, 1H), 6.55 (s, 2H), 3.94 (s, 3H), 3.86 (s, 6H), 1.88 (s, 3H), 1.66 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 186.8, 157.8, 154.5, 153.3, 142.2, 140.9, 140.2, 139.7, 139.4, 138.0, 135.0, 134.8, 133.9, 133.8, 132.7, 132.0, 130.8, 130.3, 129.5, 129.3, 128.6, 128.2, 128.1, 127.6, 120.2, 118.4, 117.7, 105.8, 61.3, 56.5, 31.9, 29.6, 29.3, 22.6, 19.1, 14.9, 14.0, 12.2.

2.3.3. BD-NOH

Compound 2 (50 mg, 0.06 mmol) dissolved in ethanol was added to a round bottle flask followed by the addition of hydroxylammonium chloride (20 mg, 0.29 mmol) and refluxed for 1 h. The reaction mixture was then washed with a brine solution and the organic solvent was removed to afford the crude mixture. Then the crude mixture was subject for column chromatography purification with eluent of ethyl acetate/ hexane (1:3, v/v) to afford a purple solid of 44 mg (86% yield). ¹H NMR (500 MHz, CDCl₃) δ: 1.62 (s, 3H), 1.72 (s, 3H), 3.85 (s, 6H), 3.93 (s, 3H), 6.56 (s, 2H), 6.71 (s, 1H), 7.04 (d, 1H), 7.45–7.28 (m, 15H), 7.53 (t, 4H), 7.71–7.66 (q, 2H), 8.23 (s, 1H). ¹³C NMR (126 MHz, CDCl₃) δ 155.4, 154.3, 150.0, 145.7, 144.4, 139.9, 139.5, 139.1, 139.0, 137.8, 137.5, 135.3, 134.9, 134.4, 134.1, 132.5, 132.3, 131.8, 130.5, 129.9, 129.7, 129.5, 129.4, 128.4, 128.1, 127.9, 127.6, 119.2, 105.5, 61.4, 56.5, 31.9, 29.7, 29.4, 22.7, 14.8, 14.1, 13.1. HRMS (ESI): Calculated for C₄₉H₄₂BF₂N₃O₄S₂ [M+H]⁺: 850.27506, Found: 850.27509.

2.4. Measurement of UV-Vis and fluorescence spectra

Methanol was used to prepare diluted stock solutions of BD-NOH (10 mM). 0.5 mM stock solutions containing ClO⁻, Cl⁻, SO₄²⁻, NO₃⁻, H₂O₂, ClO₄⁻, F⁻, were prepared by dilution using deionized water. PBS buffer and MeOH solutions for different pH at room temperature were used to perform UV/Vis and fluorescence titration experiments. The pH buffer was prepared using citric acid and potassium phosphate dibasic. A fluorescence spectrometer (λ_ex = 600 nm) was used to monitor any changes in fluorescence intensity.

2.5. The limit of detection

The limit of detection was calculated to be 1.10 μM using 5 blank solutions according to the IUPAC’s definition [27]. The limit of detection for HClO was calculated by Eq.(1)

$$Detectionlimit=3\sigma /m$$ (1)

Where σ = standard deviation of 5 blank trials while m = slope acquired from the trendline of emission intensity ratio versus concentration of HClO [28]. The Oxime probe is capable of detecting micromolar level concentrations of HClO.

2.6. Cell culture

HeLa cells were planted on a confocal dish (Mat’Tek) with a concentration of 4×10⁴ cells per confocal dish. Dulbecco’s modified Eagle’s medium (DMEM, Gibco) within an atmosphere containing 95% air and 5% CO₂ at 37 °C was used to culture HeLa cells. For CLSM Imaging, 35 mm Petri dishes are used to plate cells and then incubated with 5 μM BD-NOH in dimethyl sulfoxide (DMSO) for 30 min at 37 °C. After washing 3 times, the CLSM imaging was recorded using the cells with exciting wavelength at 560 nm. The yellow channel was set to 645±10 nm while the red channel was set at 670 ±10 nm. HeLa cells that were stained with 5 μM BD-NOH for 30 min at 37 °C presented red fluorescence. In another experiment, HeLa cells exhibited a red fluorescence while they were incubated with 5 μM BD-NOH for 30 min at 37 °C followed by 15 μM NaClO. Subsequently, the cell images were obtained at the excitation of 560 nm while the emission at 645±10 nm was collected.

3. Result and discussion

3.1. Prepare the ratiometric probe

The targeted HClO probe (BD-NOH) was synthesized via a three-step reaction sequence (Scheme 1) from the BODIPY core structure [25]. To achieve far-red absorbability, in the first step the BODIPY core was conjugated with diphenyl sulfide aldehyde using a microwave reactor to offer a 41% yield of Compound 1. After conjugation, Compound 1 exhibited an extended maximum absorption wavelength close to 650 nm. We then modified the structure of compound 1 and introduced the HClO-sensitive functional group of aldoxime via a two-step reaction. In the following step, an aldehyde group was introduced to the BODIPY scaffold at position 7 to afford compound 2. The successful synthesis of compound 2 was verified by the chemical shift peak belonging to aldehyde hydrogen at 10.11 ppm in the ¹H NMR spectrum. Lastly, compound 2 was treated with hydroxyl amine HCl salt in refluxing ethanol solution to produce the final HClO probe (BD-NOH) as a dark red solid with a yield of 86%. The disappearance of the aldehyde peak and the appearance of aldoxime hydrogen in ¹H NMR at 8.23 ppm demonstrated the successful transformation.

3.2. UV-vis and fluorescence spectra of detection for hypochlorite

BD-NOH was dissolved in the solution of PBS: MeOH = 1:3 (V/V pH = 7.8) to perform the measurement of photophysical properties, including absorbance and emission with or without HClO. As shown in the absorbance spectrum (Fig. 1), the probe revealed absorption maxima at a wavelength of 642 nm in the absence of HClO and a clear decrease in the maximum absorption peak along with a blue-shift within the presence of HClO. The quenching of absorption maxima intensity at 642 nm could be attributed to the reduced amount of BD-NOH due to continuous reaction with HClO. In addition, the altered aldoxime group after reacting with HClO could attribute to the hypochromatic shift shown in the absorption spectrum [24]. It is worthy to mention that all spectra changes were almost instant upon the introduction of HClO to the solution of the BD-NOH, showing the rapid reaction and the color change from blue to purple is obvious to naked eyes (Fig. 1 inserted picture).

Figure 1.

The absorbance spectra of BD-NOH in PBS/MeOH with the addition of HClO, the inserted picture shows the color change of the BD-NOH solution without (before, blue color) and with (after, purple color) ~1.6 equivalent of HClO.

Moreover, the aqueous solution of HClO was titrated continuously into a prepared solvent system made of a 1:3 volume ratio of PBS to MeOH (V/V, pH = 7.8) containing 50 μM of the BD-NOH. The fluorescence spectrometer (λ_ex = 600 nm) was used to record the fluorescence spectra and monitor the change in fluorescence intensity. A series of notable changes in the fluorescent intensity of BD-NOH were apparently shown with the continuous addition of HClO ions. A new emission spike positioned at 645 nm was observed, while there was only slight quenching of emission intensity at the maximum (674 nm) (Fig. 2A). Notably, more than 8-folds increase of the ratios of emission intensities at 645 and 670 nm (I₆₄₅/I₆₇₀, from 0.37 to 3.19) was observed and ascribed to the quick reaction of the oxime group towards HClO. Moreover, the ratios of emission intensities at the wavelength of 645 nm and 670 nm (I₆₄₅/I₆₇₀) were contrived as a trendline of the equivalent of HClO (Fig.2B). Significantly, the increase of the emission intensity was obvious under UV light (Fig. 2, inserted picture).

Figure 2.

Emission spectra of BD-NOH with the addition of HClO, A: emission spectra; B: ratio and trendline (R²=0.93). Inserted picture shows the color change of the HClO probe solution under UV light without (before) and with (after) 1.62 equivalent of HClO.

3.3. The selectivity of hypochlorite response towards BD-NOH

Next, we wanted to test the selectivity of this fluorescence intensity ratio change at 645 vs 670 nm. Several applicable analytes (50 equiv.) including ClO⁻, Cl⁻, SO₄²⁻, NO₃⁻, H₂O₂, ClO₄⁻, F⁻, were mixed with BD-NOH in PBS: MeOH=1:3(V/V, pH=7.8), then the fluorescence emission spectra were recorded using a fluorescence spectrometer. As shown in Figure 3, a significant change of the relative fluorescence intensity ratio at 645 nm as compared to the intensity at 670 nm was only observed with the addition of HClO. The probe displayed no significant response to the above-mentioned analytes with the exception of HClO. This demonstrated the selective response for HClO over other analytes and indicated that BD-NOH could be functional towards HClO detection in complex biological systems and possibly in living organisms.

Figure 3.

The fluorescence intensity ratio of I₆₄₅/I₆₇₀ of BD-NOH (10 μM) upon the introduction of HClO (20 μM) or various analytes (50 equiv.) in the PBS: MeOH = 1: 3 (V/V pH = 7.4) (λ_ex = 600 nm).

It is also paramount to study the HClO responding of BD-NOH with the existence of common metal ions as the control groups for the selective identification of HClO in actual bio-samples. BD-NOH was mixed with HClO along with tested metal ions, and only a slight difference in the intensity ratio (I₆₄₅/I₆₇₀) can be observed (Fig.4). It clearly indicates that BD-NOH is able to be used for the sensing of ClO⁻ in chemical, biological, and environmental analyses, where coexisting ions like several metal ions can be normally present.

Figure 4.

The fluorescence intensity ratio of I₆₄₅/I₆₇₀ of BD-NOH (10 μM) with HClO (20μM) in presence of several metal ions as background. Other metal ions (50 equivalent) in the PBS: MeOH=1:3(V/V pH=7.8) (λ_ex=600 nm).

3.4. Effect of reaction time

Time-dependent emission intensity ratios of BD-NOH with the addition of 2 equivalents of HClO in the solution were performed. As shown in Fig. 5, after several minutes, the response is still stable and can be clearly observed. This result demonstrated that the interaction with HClO is not reversible, and the reaction product is stable for long-time imaging and tracking, which makes BD-NOH an ideal fluorescence sensor for HClO detection.

Figure 5.

Time-dependent emission spectra of BD-NOH (10 μM) with the addition of HClO (20 μM), spectra were recorded after varied time intervals.

3.5. pH effects

The ratiometric responses of the oxime probe toward HClO at different pH conditions were examined (Fig. S11) with an aim to assess the probable applications of BD-NOH in diverse biological environments. Within the pH range, 4.2–7.8, comparable fluorescence intensity ratio (I₆₄₅/I₆₇₀) changes could be observed, showing that BD-NOH is capable of sensing HClO efficiently within a biological pH range. It is worthy to note that BD-NOH is not stable in basic conditions (pH > 9), as the decomposition of BD-NOH was exhibited by the fading color in basic solutions.

3.6. proposed sensing mechanism towards HClO

The proposed mechanism for the sensing of HClO with the BD-NOH was shown in Scheme 2. As presented by the prior works on oxidative reaction in the presence of hypochlorite [29], the oxime HClO-sensitive group can be oxidized by HClO to generate nitrile oxide derivative (BD-NO). After BD-NOH was mixed with HClO, the resulted mixture was subject to mass analysis, and the corresponding peak of BD-NO was identified (Figure S9). Therefore, the change from the aldoxime group to the nitrile oxide group affects the photophysical properties (both absorbance and emission) of the solution. In general, this change of functional groups can explain the blue-shifted strong emission after the introduction of HClO, which is attributed to a more potent intramolecular charge transfer (ICT) effect, considering that the electron-withdrawing capacity of the “CN=O” group is higher than that of the aldoxime group. A similar ICT mechanism has been used to explain the sensing effect of a green BODIPY based HClO sensor [22]. Furthermore, the structural change was also confirmed by the decrease of the aldoxime hydrogen peak at 8.23 ppm with the addition of HClO (Fig. S12).

Scheme 2.

Possible mechanism of BD-NOH to BD-NO with enhanced emission intensity.

3.7. Cellular imaging

To demonstrate the capacity of the probe to detect HClO in cellular conditions, studies of the cell images were conducted using a confocal microscope. To measure the performance of BD-NOH in detecting intracellular HClO levels, HeLa cells were stained with BD-NOH (5 μM), followed by the addition of NaClO (15 μM) for 30 min. Emission intensity increase from the yellow channel (λ_em= 645±10 nm) can be observed after NaClO introduction. As shown in Fig.6, the cells incubated with only BD-NOH (5 μM) for 30 min under the red channel (A1) depicted fluorescence, while images of the cells from the yellow channel (A2) depicted no fluorescence. As portrayed in Fig 6 (B1-B3), the fluorescence images correspond to the cells that were incubated with BD-NOH (5 μm) for 30 min, followed by the addition of NaClO (15 μM) for 30 min incubation. The fluorescence image in the yellow channel (B2) could be apparently noticed and the red channel (B1) looked similar compared to that of only the oxime probe (A1). These cellular experiments demonstrated that BD-NOH has decent permeability to the cell membrane, and it is capable of detecting HClO at the cellular level. More importantly, the ratiometric analysis of these fluorescence images offered a better way to track the allocation of HClO inside of cells.

Figure 6.

(A1-A3) Incubated with BD-NOH only: (A1) red channel; (A2) yellow channel; (A3) merged images; (B1-B3) Incubated with BD-NOH, and then treated with HClO: (B1) red channel; (B2) yellow channel; (B3) merged images; (C) ratio image of B2 divided by B1. Excited at 560 nm, the red and yellow channels were recorded at 670± 10 nm and 645±10 nm, respectively scale bar = 10 μm.

4. Conclusion

We extended the conjugated system of BODIPY to prolong the absorption and emission wavelengths to the far red region and developed a ratiometric HClO probe (BD-NOH) with a specific detection for HClO. The selectivity towards HClO as well as the effect of the presence of common metal ions was studied. The mechanism of detection of HClO was proposed to be the oxidation reaction of aldoxime to nitrile oxide, which structural change affect the photophysical properties. In addition, the bio-application of BD-NOH in HeLa cells was analyzed and shown in confocal fluorescence microscopy, and related ratiometric mapping of the HClO distribution demonstrated the feasibility of in vitro monitoring. Overall, the extended wavelengths of BD-NOH should allow for the application of this research to living organisms.