Two-Step Sensing, Colorimetric and Ratiometric Fluorescent Probe for Rapid Detection of Bisulfite in Aqueous Solutions and in Living Cells.

Bisulfite and sulfite (HSO3 -/SO3 2-) are not only widely used toxic chemicals but also active anions with important biological functions. Hence, the development of new detection methods for HSO3 -/SO3 2- is important for environmental security and human health. In this paper, we report a symmetrical hemicyanine for the detection of HSO3 -/SO3 2-, SHC, which is constructed with p-diphthalaldehyde with trimethylbenzoindolium via condensation. The red fluorescent probe can fast respond to HSO3 -/SO3 2- (<30 s) to give cyan fluorescence, and its sensing process is twice nucleophilic additions, which were observed from fluorescence response, initial ratiometric change, and subsequent turn-on increment; especially in a low-concentration level, the ratiometric fluorescence measurement can eliminate environmental interference. This probe can achieve a quantitative detection of HSO3 -/SO3 2- in a wide concentration range. Furthermore, the probe SHC is a mitochondria-specific probe for ratiometric fluorescent detection of HSO3 -/SO3 2- in living cells.

Introduction

Sulfur dioxide (SO2) is both a main atmospheric pollutant and a valuable commercial reagent, and human exposure to SO2 has become increasingly widespread because of the combustion of fossil fuels and the industrial manufactures such as paper pulp manufacturing and metal processing. More and more medical studies have confirmed that expose to SO2 may cause not only respiratory responses[1] but also lung cancer, cardiovascular diseases, and some neurological diseases, such as migraine headaches and brain cancer.[2]

SO2 dissolves in water to form a pH-dependent equilibrium between bisulfite and sulfite (HSO3–/SO32–) with a molar ratio of about 3:1 in neutral aqueous solution. Bisulfite and sulfite are widely used as a preservative for beverages and food to prevent oxidation and bacterial growth.[3] However, HSO3–/SO32– is toxic in high doses, which is associated with allergic reactions and food intolerance symptoms.[4] Hence, an acceptable daily intake (lower than 0.7 mg kg–1 of body weight) has been issued by the Joint FAO/WHO Expert Committee on Food Additives. The labeling of products containing no more than 10 ppm (125 μM) sulfite in foods or beverages has also been required by the U.S. Food and Drug Administration (FDA).[5]

Endogenous HSO3–/SO32– can be metabolically generated from thiol-containing amino acids, such as cysteine and glutathione.[6] The studies have showed that HSO3–/SO32– has an endothelium-dependent vasorelaxing effect at low concentrations (<450 μM) and also functions as messengers in cardiovascular systems.[7] For this reason, the development of new detection methods for HSO3–/SO32– is important for environmental security and human health. Because of the advantages of simplicity, sensitivity, nontoxicity, and ease of operation, fluorescent probes have been recognized as efficient molecular tools for visualizing anions in living systems.[8] In early years, some pioneer works about fluorescent probes for bisulfite/sulfite anion have been reviewed.[9] In recent years, some excellent fluorescent probes have been designed by various effective reactions of HSO3–/SO32– including nucleophilic addition to an aldehyde,[10] mediated levulinate cleavage,[11] nucleophilic addition to an unsaturated bond,[12] and others.[13] However, there are still some limitations in the reported probes such as poor selectivity over biothiols or H2S for most aldehyde- or levulinate-based fluorescent probes, long response time (>5 min for most probes), and high detection limits (>1 μM for half of probes). Hence, it is still a challenge to develop more reliable and quick response probes for HSO3–/SO32–. In this work, we prepared a symmetrical dual-hemicyanine ratiometric fluorescent probe SHC, which can detect HSO3–/SO32– in solutions fast and sensitively and achieve detection of HSO3–/SO32– in living cells (Scheme ).

Scheme 1

Chemical Structures and the Sensing Reaction of SHC

Results and Discussion

Synthesis of Related Compounds

Synthesis of two compounds, 2 and the probe SHC, involves the same condensation reaction using the benzindole as a starting material (Scheme ). The reaction of 2,3,3-trimethyl-1-ethylbenz[e]indolium iodide (1)[14] and excess diphthalaldehyde affords major product 2 (yield, 66%). Under the same condition, the condensation of compound 2 with compound 1 forms the probe SHC in the yield of 63%. Also, the reaction of compound 1 with diphthalaldehyde in the ratio of 2.5:1 gives directly the target product SHC. The structures of two compounds were fully characterized by 1H NMR, 13C NMR, and high-resolution mass spectroscopy (HRMS) analyses.

Scheme 2

Synthetic Procedure of the Probe SHC

(i) Ethyl iodide, toluene, 100 °C, 20 h; (ii) p-diphthalalhyde, NaOAc, acetic anhydride, under N2, 60 °C, 4 h; (iii) 1, NaOAc, acetic anhydride, under N2, 60 °C, 4 h.

Spectral Response to Bisulfite

With the two compounds in hand, we first determined their photophysical properties. Figure shows the UV/vis absorption and fluorescence spectra of 10 μM SHC or compound 2 in a phosphate buffer solution [1% dimethyl sulfoxide (DMSO), pH 7.4], a broad absorption band (330–570 nm), λmax 470 nm for SHC and 367 nm (with a shoulder at 438 nm) for 2 and fluorescence band (520–700 nm), λmax 595 nm for SHC and 586 nm for 2. After the addition of 10 equiv of NaHSO3 for 30 min, the solutions of both SHC and 2 display almost disappearance of the long-wavelength band in both the absorption and fluorescence spectra and appearance of fluorescence at the short-wavelength band with a peak at 482 nm. Obviously, fluorescence maxima of the probe SHC and the sensing product are about 595 and 482 nm, respectively. Hence, the probe SHC would exhibit a colorimetric and ratiometric fluorescence response toward HSO3–.

Figure 1

UV/vis absorption spectra (a) and fluorescence spectra [(b) λex = 345 nm] of 10 μM SHC (black) or 2 (blue) in the buffer solution (1% DMSO, pH 7.4) before and after the addition of 10 equiv NaHSO3.

To obtain more detailed information, we monitored the sensing process of SHC to HSO3–/SO32– with UV/vis absorption and fluorescence spectroscopies. After the addition of 10 equiv of NaHSO3–, the absorption spectra of 10 μM SHC in the phosphate-buffered saline (PBS) solution (1% DMSO, pH 7.4) decrease rapidly in the long-wavelength region (330–570 nm) and increase in the short-wavelength region (250–280 nm) (Figure a). The probe exhibits ratiometric fluorescent response to HSO3–, decreases and disappears in the long-wavelength region (550–680 nm), and increases in the short-wavelength region (450–550 nm) (Figure b). After the addition of HSO3– for 30 s, 90% absorption change and full disappearance of fluorescence in the long-wavelength region occur already. This implies that the probe can fast respond to HSO3–.

Figure 2

Time-dependent absorption (a) and fluorescence spectra [(b) λex = 380 nm] of SHC (10 μM, pH 7.4) upon addition of NaHSO3 (10 equiv) with 30 s intervals for 5 min.

In addition, a long-time monitoring displays excellent photo- and thermostability for both the probe and the sensing product shown in Figure S1 in the Supporting Information.

Furthermore, spectral response of SHC toward different equivalents of HSO3– (0–20 equiv) was observed by UV/vis absorption and fluorescence spectroscopies (Figure ), and more fluorescence titration data were provided in Figure S2a in the Supporting Information. As shown in Figure , the spectral changes show clearly two processes, fast decrease for the range of NaHSO3 (0–5 equiv) and slow decrease for 6–20 equiv of NaHSO3 in the long-wavelength-band absorbance (350–570 nm) (Figure a), and gradual disappearance in the long-wavelength-band fluorescence in less than 6 equiv of HSO3– and increase in the short-wavelength-band emission centered at 480 nm for the range of 6–20 equiv of NaHSO3 (Figures b and S2a,b). The two processes could imply two-step sensing reactions of the dual-hemicyanine with HSO3–, the first addition in the range of 0–5 equiv of NaHSO3 and the second addition for 6–20 equiv of NaHSO3. The fluorescence emission at 480 should be assigned to the benz[e]indol moiety. The monoadduct could have a low fluorescence efficiency (480 nm) because of the intramolecular energy transfer (ET) from the benz[e]indol moiety to the rest of the conjugation system, and the diadduct would emit more than twice as strong fluorescence as that of the monoadduct, shown in Figure (bottom).

Figure 3

Upper: UV/vis absorption (a) and fluorescence (b) spectra of 10 μM SHC solution (pH 7.4) upon additions of various amounts of NaHSO3 (0–20 equiv), incubation for 20 min, under excitation at 380 nm, and (c) plot of the ratio of fluorescence intensities (F480/F600) vs the concentration of HSO3– (0–10 μM). Bottom: the sensing reactions of SHC with HSO3–.

On the basis of the fluorescence titration, the fitting straight line was obtained from the plot of the ratio (F480/F600) of fluorescence intensities versus the concentration of NaHSO3 (0–10 μM) and the limit of detection (LOD) for SHC to HSO3– was obtained as 0.1 μM from the calculation in terms of LOD = 3δ/k (n = 11), where k is the slope of the fitting straight line between the increment versus the concentration of HSO3– and δ is the standard deviation of a blank measurement (Figure c). The plot of F480/F600 versus [NaHSO3] displays a good linear relationship because only monoaddition occurs in the concentration range of NaHSO3 (0–10 μM). Moreover, the disappearance of the fluorescence peak at 600 nm involves in the first addition (i); thus, the plot of fluorescence intensity at 600 nm versus the concentration of NaHSO3 should exhibit good linearity; besides, there will be a linear relation in the second addition (more than 5 equiv of NaHSO3). For this reason, from the plot of the fluorescence intensity at 600 nm (0–50 μM) or 480 nm (50–110 μM) versus the concentration of NaHSO3, LODs of SHC were obtained as 85 and 91 nM, respectively, shown in Figure S2c,d. These values are much lower than the standard of 10 ppm (125 μM) required by the U.S. Food and Drug Administration.[5]

At a low concentration level of HSO3–/SO32–, the ratiometric fluorescent detection can eliminate environmental interference and the detection based on turn-on fluorescence increment at 600 nm for the high-concentration level. Hence, SHC can achieve detection of HSO3–/SO32– in a wide concentration.

Sensing Mechanism

To verify the sensing mechanism, 1H NMR spectra of SHC before and after the addition of excess NaHSO3 were measured. As shown in Figure , the chemical shifts at 4.91, 2.08, and 1.59 ppm are assigned to the proton Ha, Hc, and Hb of the probe, respectively. After the addition of excess NaHSO3, these proton signals remove to high field, 1.91(c′), 1.58(a′), and 1.13(b′) ppm, and the chemical shifts of the protons (d, e) at carbon–carbon double bonds remove from the aromatic region to 4.96 ppm (d′, e′). Moreover, the formation of the single adduct SHC–HSO3 was confirmed by HRMS, where a dominant peak at a value of m/z 655.2992 corresponding to [SHC–2I– + HSO3]+ (calcd 655.2989) shown in Figure S3. Therefore, the sensing reaction was confirmed to be the nucleophilic addition of the probe SHC with HSO3–.

Figure 4

Partial 1H NMR spectra of SHC in DMSO-d6–D2O (v/v 9:1) before (black) and after (red) the addition of excess NaHSO3.

Selectivity

To evaluate the selectivity of the probe for HSO3–/SO32–, we measured the UV/vis absorption (Figure a) and fluorescence spectra (Figure b) of SHC before and after the addition of various analytes, respectively. The absorption spectra of SHC displayed a large change only in the presence of HSO3– and little change for three biothiols, Hcy, GSH and Cys, and other analytes caused no significant change shown in Figure a. For fluorescence response, only HSO3– causes a large change in the fluorescence spectra of SHC (Figure b). The fluorescence profiles at 481 nm showed a remarkable selectivity for HSO3– over other analytes including HS– and biothiols. The increment of fluorescence ratio (F481/F595) of SHC to HSO3–/SO32– is more than 200-fold, shown in Figure c.

Figure 5

UV/vis absorption (a) and fluorescence spectra (b) of SHC (10 μM) in the presence of 10 equiv of various analytes in the PBS solution (1% DMSO, pH 7.4) recorded after 20 min, and (c) ratio (F481/F595) of fluorescence intensities from (b), λex = 345 nm. (1): Blank, (2): HSO3–, (3): CO32–, (4): F–, (5): Br–, (6): Cl–, (7): I–, (8): NO2–, (9): NO3–, (10): HPO42–, (11): HS–, (12): SO42–, (13): Cys, (14): GSH, and (15): Hcy. Photos for color change (d) and fluorescence (e) of corresponding solutions.

The sensing behavior can be easily observed by the naked eyes from both the color change and fluorescence of solutions. As shown in Figure d,e, the SHC solution only to HSO3– displays a color under room light and cyan fluorescence under a portable UV lamp (365 nm). Hence, SHC reveals a high selectivity for HSO3– over other relevant analytes.

pH Effects and MTT Analysis

To assess the functions of SHC under physiological conditions, the absorption and fluorescence spectra of 5 μM SHC with and without the addition of HSO3– (5 equiv) solution were recorded at different pH values. The pH-dependent absorption and fluorescence responses of SHC to HSO3– reveal a remarkable change of absorbance and significant fluorescence enhancements under physiological conditions (pH 6–10) (Figure S4). This indicates that SHC could be used as a fluorescent probe in a biological system.

In order to detect HSO3–/SO32– in living cells, a MTT analysis was performed to assess the cytotoxicity of the probe. In the MTT assays, HepG2 cells were dealt with SHC at different concentrations from 10 to 30 μM for 24 h. The results show low toxicity to cultured cells under the experimental condition, and the cell viability is more than 90% for SHC at 10 μM (Figure S5). These data show that the probe SHC has low cytotoxicity.

Cell Imaging

Finally, the probe SHC was utilized for imaging of HepG2 cells. HepG2 cells were seeded on a 24-well plate in a culture medium for 24 h. No fluorescence was observed from nonstained HepG2 cells in both channels (Figure a–d). The HepG2 cells were incubated with SHC (10 μM) for 30 min, followed by PBS washing three times. Confocal fluorescence images exhibit no emission for the blue channel and red fluorescence for the red channel under excitation at 405 nm (Figure e–h). In contrast, after being further incubated with 0.1 mM NaHSO3 for 30 min, the HepG2 cells emit bright blue fluorescence in the blue channel and no fluorescence for the red channel (Figure i–l). This shows that the probe SHC is a permeable cell membrane and potential fluorescent probe to detect HSO3–/SO32– in living cells.

Figure 6

Confocal fluorescence images of HepG2 cells incubated without (a–d) and with 10 μM SHC for 0.5 h (e–h) and then 100 μM NaHSO3 for 0.5 h (i–l). Images were acquired using 405 nm excitation and signal collection from the blue channel (420–550 nm) and the red channel (550–700 nm).

Furthermore, cyanine derivatives could localize at mitochondria for their positive charge. To explore mitochondria localization, we performed costaining experiment with a commercial mitochondrial dye, MitoTracker Red FM. HepG2 cells were stained with SHC and the mitochondria dye in succession. As shown in Figure , the blue-channel image for SHC with HSO3– merged well with the red-channel image for the mitochondrial dye and good consistency for fluorescence intensity profiles across the line (Figure e,f). The colocalization coefficient (Pearson’s coefficient) of SHC and the mitochondria dye is 0.94 shown in Figure S6. The high overlap coefficient indicates that SHC can cumulate to detect HSO3– in the mitochondria.

Figure 7

Confocal fluorescence images of HepG2 cells costained with SHC (5 μM, 0.5 h), MitoTracker Red FM (0.5 μM, 0.5 h), and 50 μM NaHSO3 for 0.5 h successively. (a) Fluorescence image from the blue channel (420–550 nm), excitation at 405 nm, (b) fluorescence image from the red channel (665–750 nm), excitation at 633 nm, (c) bright-field images, (d) merge of (a–c), (e) merge of (a) and (b,f) fluorescence profile of a given region [yellow line in (e)].

Conclusions

In summary, we prepared a symmetrical dual-hemicyanine dye SHC for the detection of HSO3–/SO32– from the condensation of p-diphthalaldehyde with triethylindolium. The probe SHC can fast respond HSO3–/SO32– within 30 s as colorimetric and ratiometric fluorescence changes and observed conveniently by naked eyes, and LOD is as low as 85 nM. The sensing process is twice nucleophilic additions which can be observed with absorption and fluorescence spectroscopies, intimal ratiometric fluorescence, and subsequent fluorescent increment, and this can achieve detection of HSO3–/SO32– in a wide concentration range. Furthermore, cell imaging experiments reveal that SHC can detect HSO3–/SO32– mitochondria specifically in living cells. The new probe is promising to be utilized in a variety of chemical and biological applications.

Experimental Section

Materials and Instrumentation

All of the chemicals except synthesized compounds were obtained from commercial channels and were used as received without further purification. 1H and 13C NMR spectra were recorded in DMSO-d6 or CDCl3 with a Bruker AV spectrometer operating at 400 and 100 MHz, respectively, and chemical shifts were shown in parts per million using tetramethylsilane (TMS) as the internal standard. High-resolution mass spectra were obtained from a Thermo LTQ Orbitrap mass spectrometer. UV/vis absorption and fluorescence emission spectra were recorded with a UV/vis spectrometer (Shimadzu UV-2450) and a spectrofluorophotometer (Shimadzu RF-5301PC), respectively. Sample solutions for all measurements are phosphate buffer solutions (1% DMSO, pH 7.4). Water for preparing samples was purified with a Millipore system.

Synthesis of 3-Ethyl-1,1,2-trimethyl-1H-benzo[e]indol-3-ium iodide[14] (1)

1,1,2-trimethyl-1H-benzo[e] indole (400 mg, 1.9 mmol) and ethyl iodide (0.66 mL, 8.3 mmol) placed in a round-bottom flask were dissolved in 15 mL of toluene and stirred at 100 °C for 20 h. Cooling to room temperature, the solvent toluene was removed using a rotary evaporator. The obtained crude product was purified by column chromatography on silica (DCM/MeOH, v/v 20:1) to afford compound 1 (600 mg, 80%) as a black solid.

Synthesis of (E)-2-(4-Acetylstyryl)-3-ethyl-1,1-dimethyl-1H-benzo[e]indol-3-ium (2)

Compound 1 (150 mg, 0.41 mmol) and p-diphthalaldehyde (100 mg, 0.75 mmol) and sodium acetate (23 mg, 0.28 mmol) were dissolved in acetic anhydride and stirred under nitrogen atmosphere at 60 °C for 6 h. After cooling to room temperature, the rude product was collected by filtration. The rude product was washed with ethyl ether (20 mL) and purified using column chromatography (DCM/MeOH, v/v 40:1) to give compound 2 (130 mg, 66%).1H NMR (400 MHz, DMSO-d6, 25 °C, TMS): δ 10.14 (s, 1H, CHO), 8.60 (d, J = 16.8 Hz, 1H, HC=CH), 8.48 (d, J = 8.0 Hz, 3H, Ar-H), 8.33 (d, J = 3.2 Hz, 1H, Ar-H), 8.25 (d, J = 8.0 Hz, 1H, Ar-H), 8.19 (d, J = 8.8 Hz, 1H, Ar-H), 8.12 (d, J = 8.4 Hz, 2H, Ar-H), 7.92–7.75 (m, 3H, HC=CH, Ar-H), 4.93 (q, J = 7.2 Hz, 2H, CH2), 2.06 (s, 6H, CH3), 1.56 (t, J = 7.2 Hz, 3H, CH3) ppm. 13C NMR (100 MHz, CDCl3, 25 °C, TMS): δ 193.3, 182.4, 150.9, 140.1, 139.9, 138.7, 138.6, 133.9, 131.7, 131.3, 130.3, 129.1, 128.1, 127.2, 123.8, 115.3, 113.9, 79.7, 54.7, 43.5, 25.6, 14.7 ppm. FTMS + cESI: m/z calcd for C26H26NO, 354.1852 ([M – I–]+); found, 354.1843.

Synthesis of 2,2′-((1E,1′E)-1,4-Phenylenebis(ethene-2,1-diyl))bis(3-ethyl-1,1-dimethyl-1H-benzo[e] indol-3-ium)iodide (SHC)

Compound 1 (100 mg, 0.27 mmol) and compound 2 (100 mg, 0.21 mmol) were placed in a round-bottomed flask with 10 mL of acetic anhydride, and sodium acetate (47 mg, 0.57 mmol) was further added. The mixture reacts at 60 °C for 6 h under nitrogen protection. Cooling to room temperature, ethyl ether (20 mL) was added, washed, and filtrated to give a rude product. The rude product was purified using column chromatography (DCM/MeOH, v/v 40:1) to give the target compound, SHC (110 mg, 63%).1H NMR (400 MHz, DMSO-d6, 25 °C, TMS): δ 8.61 (d, J = 16.4 Hz, 2H, HC=CH), 8.47 (d, J = 8.8 Hz, 6H, Ar-H), 8.34 (d, J = 12.8 Hz, 2H, Ar-H), 8.27 (d, J = 8.0 Hz, 2H, Ar-H), 8.19 (d, J = 8.8 Hz, 2H, Ar-H), 7.92–7.77 (m, 6H, HC=CH, Ar-H), 4.93 (d, J = 6.0 Hz, 4H, CH2), 2.09 (s, 12H, CH3), 1.59 (t, J = 7.2 Hz, 6H, CH3) ppm. 13C NMR (100 MHz, CDCl3, 25 °C, TMS): δ 182.2, 151.1, 139.8, 138.7, 138.6, 133.9, 131.8, 131.4, 130.6, 129.1, 128.1, 127.2, 123.8, 114.6, 113.9, 55.4, 54.6, 25.8, 14.7 ppm. FTMS + cESI: m/z calcd for C42H42N2, 574.3337 ([M – 2I–]2+); found, 287.1663 (z = 2).

Cell Cultures and MTT Assays

HepG2 cells were seeded in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum in an atmosphere of 95% air and 5% CO2 at 37 °C for MTT analysis. The cells were placed in a 96-well plate, and added the probe SHC in different concentrations (0–30 μM), and incubated at 37 °C in an atmosphere of 95% air and 5% CO2 for 24 h. After removing the culture medium, 5 mg mL–1 (10 μL) MTT reagent in PBS was added into each well and incubated for 4 h. During this period, active mitochondria of viable cells reduce MTT to purple formazan, and unreduced MTT was discarded. The formazan precipitate in each well was dissolved by adding DMSO (150 μL) and then measured spectrophotometrically with a microplate reader at 570 nm. Finally, the cytotoxicity of each sample was expressed as the percentage of cell viability relative to the untreated cells.

Cell Fluorescence Images

HepG2 cells were seeded on the coverslips in 24-well plates and incubated in a humidified 5% CO2 atmosphere for 24 h with the complete DMEM containing 10% fetal calf serum at 37 °C DMSO solution of SHC was added to a well to give a concentration of 10 μM, and the volume ratio of the DMSO/culture medium is 1:100. After being incubated for 30 min, and then the cells were washed three times with PBS buffer. The cells were further treated with 0.1 mM NaHSO3 for 30 min, then removed the culture medium, and washed three times with PBS buffer. The cellular localization was visualized under a laser scanning confocal microscope (LSM 710 Meta, Carl Zeiss Inc., Thornwood, NY). The green fluorescence of cells was collected with 420–550 nm channel under excitation at 405, 665–750 nm channel at excitation at 633 nm.