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A TICT based two-photon fluorescent probe for bisulfite anion and its application in living cells
Accepted Manuscript Title: A TICT based two-photon fluorescent probe for bisulfite anion and its application in living cells Author: Shenglong Yu Xiuli Yang Zhonglong Shao Yan Feng Xinguo Xi Rong Shao Qingxiang Guo Xiangming Meng PII: DOI: Reference:
S0925-4005(16)30784-5 http://dx.doi.org/doi:10.1016/j.snb.2016.05.099 SNB 20258
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
31-3-2016 17-5-2016 18-5-2016
Please cite this article as: Shenglong Yu, Xiuli Yang, Zhonglong Shao, Yan Feng, Xinguo Xi, Rong Shao, Qingxiang Guo, Xiangming Meng, A TICT based two-photon fluorescent probe for bisulfite anion and its application in living cells, Sensors and Actuators B: Chemical http://dx.doi.org/10.1016/j.snb.2016.05.099 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A TICT based two-photon fluorescent probe for bisulfite anion and its application in living cells Shenglong Yu,a Xiuli Yang,b Zhonglong Shao,a Yan Feng,a Xinguo Xi,b Rong Shao,b Qingxiang Guo, a,c Xiangming Meng,*a,b
a
Department of Chemistry, Anhui University, Hefei, 230601, China.
b
Jiangsu Collaborative
Environmental
Innovation
Protection
Center
Equipments,
for
Ecological
Yancheng
Institute
Building of
Materials
and
Technology, Yancheng
224051, China c
Department of Chemistry, University of Science and Technology of China, Hefei, 230026, China.
*Corresponding author. Fax: +86-551-63861467; Tel: +86-551-63861467 E-mail address: [email protected] (Xiangming Meng).
ABSTRACT A two-photon fluorescent probe (DMPCA) for the detection of bisulfite anion in water was developed based on twisted-intramolecular-charge-transfer (TICT) mechanism. Fluorescence intensity of DMPCA increased dramatically (65 fold) upon the addition of HSO3−. The two-photon absorption cross-section of DMPCA was found to be 725 GM at 700 nm upon the addition of HSO3−. The fluorescent probe showed an excellent selectivity for bisulfite anion over other anions. The nucleophilic addition reaction mechanism with aldehyde was confirmed with NMR and MALDI-TOF. The TICT detection mechanism was confirmed by viscosity-dependent fluorescence spectra and time resolved fluorescence spectra. Moreover, DMPCA was successfully applied to the detection of bisulfite anion in HeLa cells with low cytotoxicity under two-photon excitation.
Keywords: TICT; Two-photon; Fluorescent probe; Coumarin; Bisulfite anion
1. Introduction Bisulphite (HSO3−) is essential preservative for many foods, beverages, and pharmaceutical products to prevent oxidation and bacterial growth and to also control enzymatic reactions during production and storage [1–3]. Moreover, bisulfite has harmful effects on tissue, cells and biomacromolecules. It can cause visible damage such as necrosis, inhibit cell division, induce micronucleus and lead to cell death [4-6]. Therefore, the development of sensitive, selective and low cost methods for the detection of bisulfite is highly needed. Convenient tools, such as optical sensors [7, 8] and chromoreactands [9] have attracted significant research interest during last years. However, fluorescent probes provide the optimal choice to detect biological agents due to its high sensitivity, simplicity for implementation, real-time detection, and good compatibility for bio-samples [10-12]. So far, numbers of small-molecule fluorescent probes for specific detection of bisulfite have been reported [13-21]. However, most of these probes are only applicable in half-water conditions, and they are almost designed on single-photon fluorescence technology. These probes with one-photon microscopy (OPM) require excitation with short-wavelength light (ca. 350–550 nm) that limits their application in deep-tissue imaging, owing to the shallow penetration depth as well as to photo-bleaching, photo-damage, and cellular auto fluorescence. Recently,
two-photon
fluorescence (TPF) probes, which can be excited by two-photon absorption in the NIR wavelength, provided an opportunity to overcome the problems originated from the single-photon fluorescence technology [22-28]. Herein, we report a novel TICT based two-photon fluorescent probe (DMPCA) based on coumarin skeleton (Scheme 1) [29-31]. Coumarin was used as the fluorescent group for their excellent optical properties. N,N-dimethyl group(-N(CH3)2), a typical TICT donor [32-37], was
use as the electron donor. Based on previous results [38-40], phenyl alkynyl was introduced at the 7-position of coumarin to achieve good two-photon fluorescence performance. Aldehydes can selectively react with bisulfite to form aldehyde-hydrogen sulfite adduct [41, 42]. We deem that the aldehyde group of DMPCA can react with bisulfite with high selectivity and cause the change of fluorescence of the probe. Thus, the fluorescent detection signal of bisulfite could be achieved. To the best of our knowledge, DMPCA is the first TICT based two photon fluorescent probe for bisulfite.
Scheme 1. The proposed TICT mechanism for the determination of HSO3−.
2. Experimental section 2.1 General procedures All reagents and solvents were commercially purchased. All reactions were magnetically stirred and monitored by thin layer chromatography (TLC). The 1H NMR (400 MHz) and
13
C NMR (100 MHz) spectra were recorded on a Bruker Avance
spectrometer using DMSO-d6 and CDCl3 as the solvents. Fluorescence spectra were obtained using a HITACHIF-2500 spectrometer. UV-vis absorption spectra were recorded on a Tech-comp UV 1000 spectrophotometer. MS spectra were conducted by Bruker autoflex III MALDI TOF mass spectrometer. The test solution of probe DMPCA (10μM) in 0.2 M Na2HPO4 citric acid (pH 5.0 aqueous buffer containing 1% DMSO) was prepared. The fluorescence quantum yields were detected by HORIB FluoroMax-4P. The two-photon cross section was tested in DMSO with 1mM DMPCA+NaHSO3. TPEF spectra were measured using femtosecond laser pulse and Ti: sapphire system (680–1080 nm, 80 MHz, 140 fs, Chameleon II) as the light source. 2.2 Preparation of the Test Solution
One millimole of inorganic salt (NaHSO3, NaNO3, Na2CO3, NaHSO4, NaF, NaCl, NaCN, KBr, KSCN, KI, Cys·HCl, NaHS and Na2S2O3) was dissolved in distilled water (10 mL) to afford 1×10−1mol/L aqueous solution. The stock solutions were diluted to desired concentrations with water when needed. The resulting solution was shaken well and incubated for 5 min at room temperature before recording the spectra. 2.3 Synthesis of probe DMPCA The DMPCA was easily synthesized via four steps from readily available initial materials with an overall yield of 13.0% (Scheme 2). The structures of DMPCA and intermediates were all confirmed by 1H NMR and 13C NMR.
Scheme 2. Synthetic route of DMPCA.
Compound 1. 3-Iodophenol (10g, 45mmol) was dissolved in anhydrous acetonitrile (160ml), cooled in an ice bath and magnesium chloride (12.8g, 134mmol) added portion-wise over 10 min. Triethylamine (25.3ml, 363mmol) was added to this mixture over 5min, followed by portion-wise addition of paraformaldehyde (5.47g, 636mmol). After complete addition the mixture was heated at reflux for 18.5 hours. The mixture was cooled and quenched by the addition of sat. NH4Cl (350ml) and extracted with EtOAc (3xl50ml). The combined EtOAc layers were washed with sat. NaHCO3 (2x150mml), 1N HCl (2 x 150ml), and sat. NaCl (2x100ml), dried over Na2SO4, filtered and evaporated. The residue was purified by MPLC on silica gel eluting with a gradient rising from 100% hexanes to 20% EtOAc in hexanes. Product containing fractions were combined and evaporated and crystallized from hot hexanes to give of 5.64g the compound 1 (25.5mmol, 50% ). 1H NMR(400 MHz, CDCl3,ppm) : 11.01 (s, 1H),
9.85 (s, 1H), 7.44 (s, 1H), 7.39 (d, J =8.1 Hz, 1H), 7.23 (d, J =8.1 Hz, 1H). 13C NMR (100MHz, CDCl3, ppm): δ 196.12 , 161.31, 134.22 , 129.42 , 127.26 , 119.95 , 105.18. Compound 2. Mixture of 2-hydroxy-4-iodobenzaldehyde (20.31g, 81.89mmol), propionic anhydride (20.99g, 230.5mmol), and K2CO3 (0.5528g, 4mmol) was refluxed until the color of the reaction mixture became brown (16 h). The excess of propionic anhydride was distilled off. The residue was poured into crushed ice, and the pH was adjusted to 7 using NaHCO3. The precipitate was filtered off, washed with water, dried, and recrystallized from a mixture of hexane and ethyl acetate (4:1) to give 12.88g of the compound 2 (45.04mmol, 55%) as white needles. 1H NMR(400 MHz, CDCl3,ppm) :7.64 (s, 1H), 7.55 (d, J=8.1 Hz, 1H), 7.44 (s, 1H), 7.11 (d, J=8.1 Hz, 1H), 2.17 (s, 3H). 13C NMR (100MHz, CDCl3, ppm): δ 161.34 , 153.19, 138.63, 133.52, 127.97, 126.64 , 125.57 , 119.03, 95.44, 17.37. Compound 3. A mixture of the compound 2 (0.89g, 3.1mmol) and SeO2 (0.69 g, 6.2mmol) was ground in a mortar and transferred into a microwave vial, which was sealed and irradiated at 150W and 170oC for 1h. Caution! An extraction funnel was situated over the reaction vial during the reaction with SeO2 to remove noxious vapor. The product was then extracted with EtOAc, concentrated, and purified by flash chromatography to afford 0.48g brown crystals of the compound 3 (1.61mmol, 52%). 1H NMR(400 MHz, CDCl3, ppm) : 10.22 (s, 1H), 8.36 (s, 1H), 7.80 (s, 1H), 7.71 (d, J=8.1 Hz, 1H), 7.37 (d, J=8.2 Hz, 1H). 13C NMR (100MHz, CDCl3, ppm): δ 187.39 , 159.32, 155.15 , 144.90, 134.84 , 131.32 , 126.60 , 122.23, 117.63, 102.05. DMPCA. The mixture of compound 3 (5.43g, 18.1mmol), 4-ethynyl-N,N-dimethylaniline (3.15g, 21.72mmol), PdCl2(PPh3)2 (71.8 mg, 0.905mmol), Et3N (3.66 g, 36.2mmol), and THF (20 mL) was heated at 30oC for 3h. After cooling to room temperature, the mixture was filtered to
remove salts and followed with the addition of 100 mL H2O. The resulting mixture was extracted with EtOAc (50 mL×3). The organic phase was combined and dried with anhydrous Na2SO4, and evaporated to yield a crude product that was recrystallized in EtOAc/PE to give 5.22g of DMPCA (16.48mmol, 91%). 1H NMR (400MHz, CDCl3, ppm) : 10.25 (s, 1H), 8.37 (s, 1H), 7.60 (d, J=8.0 Hz, 1H), 7.43 (t, J=8.7 Hz, 4H), 6.67 (d, J=8.3 Hz, 2H), 3.03 (s, 6H). 13C NMR (100MHz, CDCl3, ppm): δ 187.73, 159.95, 155.44, 150.80, 144.77, 133.27, 131.77, 130.27, 127.93, 120.94, 118.53, 117.03, 111.74, 108.33, 98.30, 87.17, 40.04. MALDI-TOF MS spectrum: [DMPCA-H]- (m/zcalcd =316.11). 2.4 Measurement of Two-photon Absorption Cross-section (ä) Two-photon
excitation
fluorescence
(TPEF)
spectra
were
measured
using
femtosecond laser pulse and Ti: sapphire system (680~1080 nm, 80 MHz, 140 fs, Chameleon II) as the light source. All measurements were carried out in air at room temperature. TPA cross sections were measured using two-photon-induced fluorescence measurement technique. The TPA cross-sections (δ) were determined in acetonitrile with 1mM DMPCA+NaHSO3 by comparing their TPEF according to the following equation:
ref
ref cref nref F c n Fref
Here, the subscripts ref stands for the reference molecule. δ is the TPA cross-section value, c is the concentration of solution, n is the refractive index of the solution, F is the TPEF integral intensities of the solution emitted at the exciting wavelength, and Φ is the fluorescence quantum yield. The δref value of reference was taken from the literature [43]. 2.5 Cell culture and two-photon fluorescence microscopy imaging
For two-photon bio-imaging, HeLa cells were cultured in DMEM supplemented with 10% FCS, penicillin (100μg/mL), and streptomycin (100μg/mL) at 37 oC in a humidified atmosphere with 5% CO 2 and 95% air. Cytotoxicity assays show that DMPCA is safe enough for two-photon bio-imaging at low concentrations, so that the cells were incubated with 10μM DMPCA at 37oC under 5% CO2 for 30 min, washed once and bathed in DMEM containing no FCS prior to imaging and/or HSO3- addition. Then 100μM HSO3was added in the growth medium for 0.5 h at 37 oC, washed 3 times with PBS buffer. Then, cells were imaged on a confocal microscope (Zeiss LSM 510 Meta NLO). Two-photon fluorescence microscopy images of labeled cells were obtained by exciting the probe with a mode-locked titanium-sapphire laser source set at wavelength 700 nm.
3. Results and discussion As shown in Scheme 2, the probe DMPCA was synthesized via four steps from 3-Iodophenol. The structures of DMPCA and the intermediates were all confirmed by 1H-NMR and
13
C-NMR. The structure of DMPCA was also confirmed by X-ray crystallography analysis
(CCDC 1401985). The single crystals of DMPCA grew from anhydrous ether–petroleum ether (60–90 °C) solution. The crystal structure of DMPCA was depicted in Fig. 1. The parameters associated with the crystal data are shown in Table 1.
Fig. 1. Molecular structure of DMPCA (ORTEP drawings of DMPCA showing thermal ellipsoids at 30% probability level).
Table 1. X-ray crystallography data for DMPCA.
3.1 UV–vis and fluorescence spectra study Fig. 2 shows the change of the fluorescence spectra of DMPCA (10uM in 0.2M citric acid buffer, pH=5.0, λex =345 nm) upon the addition of HSO3−. Upon the increasing of HSO3− concentration, the initial fluorescence intensity at 415 nm increased linearly with 65 fold and reached a plateau when 10 equiv. of HSO3− was added, as show in Fig. S1, and the limit of detection (LOD) was calculated to be 1.38 10-7μM (σ=0.093, k=2.02, R2=0.994). In addition, Fig. S2 shows the change on the UV-vis spectrum when HSO3− was added to the Na2HPO4 citric acid buffer (0.2 M, pH 5.0) solution containing probe DMPCA (10μM). Upon the addition of HSO3−, the absorption peaks of DMPCA at 345 nm was increased clearly, which may indicate the formation of new compound.
Fig. 2. Emission spectral changes for the probe DMPCA (10μM) (λex= 345 nm, λem = 415 nm) in 0.2 M Na2HPO4 citric acid (pH 5.0 aqueous buffer containing 1% DMSO) on gradual addition of HSO3− (0–140μM). Each spectrum was recorded 5 min after HSO3− addition. Insert: The relationship between the fluorescence intensity (F415nm) and the bisulfite concentrations. Working conditions: pH 5.0, Reaction time = 5 min.
3.2 The pH stability The effect of pH on the fluorescence properties of the system was investigated (Fig. 3). It is obvious that the fluorescent signal of the probe is stable within pH scale 3.2-9.3. However, the fluorescence signal of the probe with bisulfite anion increased from pH 3.2 to 5.0, and decreased at pH values above 6.0. The aldehyde-bisulfite adduct is stable within the pH 5.0 to 6.0. These result is similar to previous reported bisulfite probes [44], thus, the pH 5.0 buffer solution was selected as experimental detection condition.
Fig. 3. pH effect on the emission intensity of DMPCA. Bisulfite concentration: (a) 0 and (b) 100μM.
3.3. Response time The response time of DMPCA to bisfute was also studied in Na2HPO4/citric acid buffer (0.2 M, pH 5.0) solution. As shown in Fig. 4, we can see that the addition reaction between DMPCA and bisfulite can be finished within 2 minutes which is very rapid compared with other bisulfite probes [45, 46]. The fast response time makes DMPCA applicable in real detection of bisulfite.
Fig. 4. The response time of DMPCA(10uM) to bisulfite. Bisulfite concentration: (a) blank and (b) 100μM, detection condition: pH=5.0.
3.4 Selectivity over other anions Ion selectivity study was performed in Na2HPO4 citric acid buffer (0.2 M, pH 5.0) solution. The optical responses of DMPCA (10μM) to various anions were investigated by the emission spectroscopy. As shown in Fig. 5, none of the anions except HSO3− induced the change of the fluorescence. The color change of fluorescence DMPCA to various ions was shown in Fig. S3, it’s obvious that only HSO3− turn on the blue fluorescence of the solution of DMPCA. Competitive experiments were also carried out using HSO3− (100μM) mixed with other anions (200μM). As shown in Fig. S4, other anions present little impacts on the detecting signal (I415nm) of DMPCA. These results suggest that DMPCA has good selectivity for HSO3− over other anions. Fig. 5. Emission spectra of the probe (10μM) with various anions (200μM except HSO3- 100μM), the emission spectra was recorded after 5 min. Detection condition: pH=5.0.
3.5. Nucleophilic addition mechanism To analyze the reaction mechanism, H-NMR and MS titration was carried out. Form 1H NMR spectrum, we can see that the aldehyde proton at around δ 10.25 was dramatically shifted to 4.79 (Fig. 6). These results indicated that the aldehyde group were reacted with bisulfite ions under detection condition. The reaction mixture of DMPCA with NaHSO3 was characterized by MALDI-TOF MS spectrum. As shown in Fig. 7, DMPCA revealed a main peak at 315.88 before the addition of NaHSO3, corresponding to the species [DMPCA-H]- (m/zcalcd =316.11). After the addition of NaHSO3, a relatively weak peak at about 420.13 appeared coinciding exactly with that for the adduct species [DMPCA+NaHSO3-H]- (m/zcalcd = 420.06). The above data suggested that the nucleophilic addition reaction of the probe’s formyl group and the bisulfate ion was the origin of the detection signal. .
Fig. 6.The proposed mechanism for the detection of HSO3−, The 1H-NMR spectra of the DMPCA (top) and DMPCA+10eq. NaHSO3 (bottom) in DMSO-d6.
Fig. 7. (a) MALDI-TOF MS spectrum of DMPCA (DCTB as matrix). (b) MALDI-TOF MS spectrum of DMPCA+ NaHSO3 (DCTB as matrix).
3.6. The viscosity-dependent fluorescence spectra and fluorescence lifetime To further understand the TICT mechanism of DMPCA, the viscosity-dependent fluorescence emission was carried out in the water-polyethylene glycol system. As shown in Fig. 8(a), with the increase of the solvent viscosity, the significant fluorescence enhancement (about
30-folds) was observed. Increased solvent viscosity would prohibit the rotation of the C-C bond of DMPCA (as shown in scheme 1), thereby prevent the non-radiative energy loss of the excited singlet state. As a result, the fluorescence emission was increased subsequently. On the other hand, fluorescence lifetime of DMPCA with and without bisulfate ion was detected by time resolved fluorescent spectra. As shown in Fig. 8(b) and Table S1, the fluorescence decay curve of DMPCA was biexponential (τ1=0.65 ns, τ2=2.74 ns), in which the slow component (τ2=2.74 ns, 33%) was attributed to the TICT state. However, upon addition of bisulfate ion the fluorescence decay curve of DMPCA was changed into monoexponential (τ=1.08 ns) due to the disappearance of TICT state. Thereby, the fluorescence emission was increased after HSO3- addition. These results indicated that the change of spectra of DMPCA was attributed to TICT process.
Fig. 8 (a) Fluorescence emission spectra of DMPCA (10μM) in water-polyethylene glycol 400 system with increased viscosity, λex=345 nm. (b) Fluorescence decay curves for DMPCA and DMPCA +HSO3- in water.
3.7. Two-photon absorption cross-sections The two-photon absorption cross-sections of DMPCA+HSO3- were detected using the two-photon induced fluorescence measurement spectra. As shown in Fig. 9, the maximum two-photon absorption cross-section (δmax) value of DMPCA+HSO3- is 725 GM at 700 nm (Φ=0.07). The large two-photon absorption cross-sections of DMPCA+HSO3- suggested that probe DMPCA would be a good two-photon probe for bisulfite ion.
Fig. 9. Two-photon cross-sections of DMPCA+HSO3-.
3.8 Cell cytotoxicity and fluorescence microscopy imaging Cell cytotoxicity assays were conducted using HeLa cells to test the cytotoxicity of DMPCA. The cell viability remains more than 95% after treated with 10μM DMPCA for 24 h (Fig. S5). The low cytotoxicity of DMPCA confirmed that the probe is safe for bio-imaging. With the above data in hand, we finally investigated the utility of DMPCA to image HSO3− in living cells under one-photon and two-photon excitation. HeLa cells were cultured and stained with DMPCA (10μM) within 30 min and washed by PBS buffer and then treated with HSO3− (100μM) for another 30 min. TPM images were obtained by exciting the probe at wavelength 700 nm. The blue emission channel was chosen as the detecting windows for DMPCA and DMPCA+HSO3−. As shown in Fig. 10, before the addition of HSO3−, the blue emission channel exhibited no fluorescence. After HSO3− addition, the fluorescence of the blue emission channel exhibited strong fluorescence dramatically. This indicated that DMPCA is cell permeable. The fluorescence images generated from the blue emission channel demonstrated that DMPCA can offer a simple and visible way to detect HSO3- in vivo in real time under two-photon excitation.
Fig. 10. (a) Bright-field image of HeLa cells incubated with 10μM DMPCA after 30 min of incubation, washed with PBS buffer, λex =700 nm (fluorescence at the blue emission channel); (b) Two-photon image of HeLa cells; (c) The overlay of (a) and (b); (d) Bright-field image of HeLa cells following a 30 min treatment with NaHSO3 (100μM, fluorescence at the blue emission channel); (e) Two-photon image of HeLa cells; (f) The overlay of (d) and (e).
4. Conclusions
In conclusion, we developed a TICT based two-photon fluorescent probe (DMPCA) for the detection of bisulfite anion in water. Upon the addition of bisulfite anion, the probe displayed obvious fluorescence enhancement (about 65 folds). Taking advantage of the nucleophilic addition reaction with aldehyde, the probe has exhibited a sensitive and selective response to the bisulfite anion against other anions. DMPCA shows maximum two-photon absorption cross-sections (725 GM) at 700nm upon the addition of bisulfite anion. Furthermore, the probe was successfully applied to the detection of the bisulfite anion in living cells under two-photon excitation.
Acknowledgements This work was supported by National Natural Science Foundation of China (21102002, 21272223, 21372005), The research fund of Jiangsu Collaborative Innovation Center for Ecological Building Materials and Environmental Protection Equipments and Anhui University.
Biographies Shenglong Yu is studying for master degree in Anhui University. He received his bachelor of science in chemistry from the department of chemistry at Anhui University.
XiuLi Yang
is an assitant professor at Jiangsu Collaborative
Innovation Center
for Ecological Building Materials and Environmental Protection Equipments and Yancheng Institute of Technology. Her research focus on the design and synthesis of functional Metal-Organic
Frameworks for sensing application.
Zhonglong Shao is an associate professor in the department of chemistry at Anhui University. His current research interests are chemosensors and theoretical calculation.
Yan Feng is an associate professor in the department of chemistry at Anhui University. Her current research interests are chemosensors and organometallic chemistry.
Xinguo
Xi
is
a
professor
at
Ecological Building Materials and Insti-tute
of
Technology.
His
Jiangsu
Collaborative
Environmental research
focus
Innovation
Protection Equipments on
the
design
Center and
and
for
Yancheng
synthesis
of
functional Metal-Organic Frameworks for sensing and catalysis application.
Rong Shao
is a
professor
at
Ecological Building Materials and Institute
Jiangsu
Collaborative
Environmental
Innovation
Protection Equipments
Center and
of
for
Yancheng Technology.
His research interest lies in chemical environmental protection and bio-chemical.
Qiangxiang Guo is a professor in department of chemistry at University of Science and technology of China and joined the department of chemistry at Anhui University as a distinguished professor, in 2015. His research interest is physical organic chemistry.
Xiangming Meng is an associate professor in the department of chemistry at Anhui University. His current research interests are chemosensors and nanoclusters.
References [1] H. Tian, J. Qian, Q. Sun, H. Bai, W. Zhang, Colorimetric and ratiometric fluorescent detection of sulfite in water via cationic surfactant-promoted addition of sulfite to α, β-unsaturated ketone, Analytica chimica acta 788 (2013) 165-170. [2] Y. Sun, S. Fan, S. Zhang, D. Zhao, L. Duan, R. Li, A fluorescent turn-on probe based on benzo [e] indolium for bisulfite through 1,4-addition reaction, Sensors and Actuators B: Chemical 193 (2014) 173-177. [3] G. Xu, H. Wu, X. Liu, R. Feng, Z. Liu, A simple pyrene-pyridinium-based fluorescent probe for colorimetric and ratiometric sensing of sulfite, Dyes and Pigments 120 (2015) 322-327. [4] W. Xu, C.L. Teoh, J. Peng, D. Su, L. Yuan, Y.T. Chang, A mitochondria-targeted ratiometric fluorescent probe to monitor endogenously generated sulfur dioxide derivatives in living cells, Biomaterials 56 (2015) 1-9. [5] J. Xu, J. Pan, X. Jiang, C. Qin, L. Zeng, H. Zhang, J.F. Zhang, A mitochondria-targeted ratiometric fluorescent probe for rapid, sensitive and specific detection of biological SO2 derivatives in living cells, Biosensors and Bioelectronics 77 (2016) 725-732. [6] X. Cheng, H. Jia, J. Feng, J. Qin, Z. Li, “Reactive” probe for hydrogen sulfite: Good ratiometric response and bioimaging application, Sensors and Actuators B: Chemical 184 (2013) 274-280. [7] S.S.M. Hassan, M.S.A. Hamza, A.H.K. Mohamed, A novel spectrophotometric method for batch and flow injection determination of sulfite in beverages, Analytica Chimica Acta 570 (2006) 232–239; [8] P.D. Tzanavaras, E. Thiakouli, D.G. Themelis, Hybrid sequential injectionflow injection manifold for the spectrophotometric determination of total sulfite in wines using o-phthalaldehyde and gas-diffusion, Talanta 77 (2009) 1614–1619. [9] G.J. Mohr, A chromoreactand for the selective detection of HSO3− based on the reversible bisulfite addition reaction in polymer membranes, Chemical Communications (2002) 2646–2647. [10] C. Huang, T. Jia, M.F. Tang, Q. Yin, W.P. Zhu, Y.F. Xu, X.H. Qian, et al. Selective and Ratiometric Fluorescent Trapping and Quantification of Protein Vicinal Dithiols and in Situ Dynamic Tracing in Living Cells, Journal of the American Chemical Society 136 (2014)
14237–14244. [11] Y.C. Chen, C.C. Zhu, Z.H. Yang, J.J. Chen, Y.F. He, J.J. Cen, Z.J. Guo, A ratiometric fluorescent probe for rapid detection of hydrogen sulfide in mitochondria, Angewandte Chemie 125 (2013) 1732–1735. [12] A.T. Wrobel, T.C. Johnstone, A. Deliz Liang, S.J. Lippard, P. Rivera-Fuentes, A fast and selective near-infrared fluorescent sensor for multicolor imaging of biological nitroxyl (HNO), Journal of the American Chemical Society 136 (2014) 4697-4705. [13] M.G. Choi, J. Hwang, S. Eor, S.K. Chang, Chromogenic and fluorogenic signaling of sulfite by selective deprotection of resorufin levulinate, Organic letters 12 (2010) 5624-5627. [14] G. Li, Y. Chen, J. Wang, Q. Lin, J. Zhao, L. Ji, H. Chao, A dinuclear iridium (III) complex as a visual specific phosphorescent probe for endogenous sulphite and bisulphite in living cells, Chemical Science 4 (2013) 4426-4433. [15] J. Chao, Y. Zhang, H., Wang, Y. Zhang, F. Huo, C. Yin, Y. Wang, Fluorescent Red GK as a fluorescent probe for selective detection of bisulfite anions, Sensors and Actuators B: Chemical 188 (2013) 200-206. [16] Y.Q. Sun, J. Liu, J. Zhang, T. Yang, W. Guo, Fluorescent probe for biological gas SO2 derivatives bisulfite and sulfite, Chemical Communications 49 (2013) 2637-2639. [17] H. Tian, J. Qian, Q. Sun, C. Jiang, R. Zhang, W. Zhang, A coumarin-based fluorescent probe for differential identification of sulfide and sulfite in CTAB micelle solution, Analyst 139 (2014) 3373-3377. [18] M.Y. Wu, K. Li, C.Y. Li, J.T. Hou, X.Q. Yu, A water-soluble near-infrared probe for colorimetric and ratiometric sensing of SO2 derivatives in living cells, Chemical Communications 50 (2014) 183-185. [19] L. Zhu, J. Xu, Z. Sun, B. Fu, C. Qin, L. Zeng, X. Hu, A twisted intramolecular charge transfer probe for rapid and specific detection of trace biological SO2 derivatives and bio-imaging applications, Chemical Communications 51 (2015) 1154-1156. [20] X. Dai, T. Zhang, Z.F. Du, X.J. Cao, M.Y. Chen, S.W. Hu, ... & B.X. Zhao, An effective colorimetric and ratiometric fluorescent probe for bisulfite in aqueous solution, Analytica chimica acta 888 (2015) 138-145.
[21] D.P. Li, Z.Y. Wang, X.J. Cao, J. Cui, X. Wang, H.Z. Cui, ... & B.X. Zhao, A mitochondria-targeted fluorescent probe for ratiometric detection of endogenous sulfur dioxide derivatives in cancer cells, Chemical Communications 52 (2016) 2760--2763. [22] Q. Xu, C.H. Heo, G. Kim, H.W. Lee, H.M. Kim, J. Yoon, Development of Imidazoline-2-Thiones Based Two-Photon Fluorescence Probes for Imaging Hypochlorite Generation in a Co-Culture System, Angewandte Chemie 127 (2015) 4972-4976. [23] S.K. Bae, C.H. Heo, D.J. Choi, D. Sen, E. H. Joe, B.R. Cho, H.M. Kim, A ratiometric two-photon fluorescent probe reveals reduction in mitochondrial H2S production in Parkinson’s disease gene knockout astrocytes, Journal of the American Chemical Society 135 (2013) 9915-9923. [24] H. Zhang, J.L. Fan, J.Y. Wang, S.Z. Zhang, B.R. Dou, X.J. Peng, An Off–On COX-2-Specific Fluorescent Probe: Targeting the Golgi Apparatus of Cancer Cells, Journal of the American Chemical Society 135 (2013) 11663-11669. [25] F. Miao, W.J. Zhang, Y.M. Sun, R.Y. Zhang, Y. Liu, F.Q. Guo, X.Q. Yu, Novel fluorescent probes for highly selective two-photon imaging of mitochondria in living cells, Biosensors and Bioelectronics 55 (2014) 423-429. [26] L.Y. Zeng, S.Y. Chen, T. Xia, C. Zhong, Z.H. Liu, Designing two-photon fluorescent probes based on the target-induced enhancement of the absorption cross-section, Chemical Communications 50 (2014) 11139-11142. [27] L.Y. Zhou, X.B. Zhang, Q.Q. Wang, Y.F. Lv, G.J. Mao, A.L. Luo, Y.X. Wu, Y. Wu, J. Zhang, W.H. Tan, Molecular Engineering of a TBET-Based Two-Photon Fluorescent Probe for Ratiometric Imaging of Living Cells and Tissues, Journal of the American Chemical Society 136 (2014) 9838-9841. [28] L. Yuan, W.Y. Lin, H. Chen, S.S. Zhu, L.W. He, A Unique Family of Rigid Analogues of the GFP Chromophore with Tunable Two-Photon Action Cross-Sections for Biological Imaging, Angewandte Chemie International Edition 52 (2013) 10018-10022. [29] B. Chen, Y. Ding, X. Li, W. Zhu, J. P. Hill, K. Ariga, & Y. Xie, Steric hindrance-enforced distortion as a general strategy for the design of fluorescence “turn-on” cyanide probes, Chemical Communications 49 (2013) 10136-10138.
[30] Y. Xie, Y. Ding, X. Li, C. Wang, J.P. Hill, K. Ariga, ... & W. Zhu, Selective, sensitive and reversible
“turn-on”
fluorescent
2′-dipyridylaminoanthracene–Cu2+
cyanide
ensembles, Chemical
probes
based
Communications
on 48
2, (2012)
11513-11515. [31] Y. Ding, Y. Tang, W. Zhu, & Y. Xie, Fluorescent and colorimetric ion probes based on conjugated oligopyrroles, Chemical Society Reviews 44 (2015) 1101-1112. [32] Y. Ding, W.H. Zhu, & Y. Xie, Development of Ion Chemosensors Based on Porphyrin Analogues, Chemical Reviews (2016). [33] S. I. Reja, I. A. Khan, V. Bhalla, M. Kumar, A TICT based NIR-fluorescent probe for human serum albumin: a pre-clinical diagnosis in blood serum, Chemical Communications 52 (2016) 1182-1185. [34] V. Tharmaraj, K. Pitchumani, An acyclic, dansyl based colorimetric and fluorescent chemosensor for Hg (II) via twisted intramolecular charge transfer (TICT), Analytica Chimica Acta 751 (2012) 171–175. [35] J. S. Yang, C. K. Lin, A. M. Lahoti, C. K. Tseng, Y. H. Liu, G. H. Lee, S. M. Peng, Effect of Ground-State Twisting on the trans→ cis Photoisomerization and TICT State Formation of Aminostilbenes , the Journal of Physical Chemistry A 113 (2009) 4868–4877. [36] M. Cigáň, J. Donovalová, V. Szöcs, J. Gašpar, K. Jakusová, A. Gáplovský, 7-(Dimethylamino) coumarin-3-carbaldehyde and its phenylsemicarbazone: TICT excited state modulation, fluorescent H-aggregates, and preferential salvation, the Journal of Physical Chemistry A 117 (2013) 4870−4883. [37] X.J. Wei, X.L. Yang, Y. Feng, P. Ning, H.Z. Yu, M.Z. Zhu, X.G. Xi, Q.X. Guo, X.M. Meng, A TICT based two-photon fluorescent probe for cysteine and homocysteine in living cells, Sensors and Actuators B: Chemical 231 (2016) 285−292. [38] H.J. Yin, B.C. Zhang, H.Z. Yu, L. Zhu, Y. Feng, M.Z. Zhu, X.M. Meng, Two-Photon Fluorescent Probes for Biological Mg2+ Detection Based on 7-Substituted Coumarin, The Journal of organic chemistry 80 (2015) 4306-4312. [39] X.M. Meng, S.X. Wang, Y.M. Li, M.Z. Zhu, Q.X. Guo, 6-Substituted quinoline-based ratiometric two-photon fluorescent probes for biological Zn2+ detection, Chemical Communications 48 (2012) 4196-4198.
[40] L. Yuan, W. Lin, Z. Cao, J. Wang, B. Chen, Development of FRET-Based Dual-Excitation Ratiometric Fluorescent pH Probes and Their Photocaged Derivatives, Chemistry-A European Journal 18 (2012) 1247-1255. [41] L.C. de Azevedo, M.M. Reis, L.F. Motta, G.O.D. Rocha, L.A. Silva, J.B. de Andrade, Evaluation of the formation and stability of hydroxyalkyl sulfonic acids in wines, Journal of agricultural and food chemistry 55 (2007) 8670-8680. [42] D.P. Kjell, B.J. Slattery, M.J. Semo, A novel, nonaqueous method for regeneration of aldehydes from bisulfite adducts, The Journal of organic chemistry 64 (1999) 5722-5724. [43] O. Varnavski, T. Goodson, L. Sukhomlinova, R. Twieg, Ultrafast Exciton Dynamics in a Branched Molecule Investigated by Time-Resolved Fluorescence, Transient Absorption, and Three-Pulse Photon Echo Peak Shift Measurements, Journal of Physical Chemistry B 108 (2004) 10484-10492. [44] Y. Yang, F. Huo, J. Zhang, Z. Xie, J. Chao, C. Yin, X. Yan, A novel coumarin-based fluorescent probe for selective detection of bissulfite anions in water and sugar samples, Sensors and Actuators B: Chemical 166 (2012) 665-670. [45] C. Wang, S. Feng, L. Wu, S. Yan, C. Zhong, P. Guo, X. Zhou, A new fluorescent turn-on probe for highly sensitive and selective detection of sulfite and bisulfite, Sensors and Actuators B: Chemical 190 (2014) 792-799. [46] W. Chen, Q. Fang, D. Yang, H. Zhang, X. Song, J. Foley, Selective, Highly Sensitive Fluorescent Probe for the Detection of Sulfur Dioxide Derivatives in Aqueous and Biological Environments, Analytical chemistry 87 (2014) 609-616.
Figures and schemes
Scheme 1. The proposed TICT mechanism for the determination of HSO3−.
Scheme 2. Synthetic route of DMPCA.
Fig. 1. Molecular structure of DMPCA (ORTEP drawings of DMPCA showing thermal ellipsoids at 30% probability level).
Fig. 2. Emission spectral changes for the probe DMPCA (10μM) (λex= 345 nm, λem = 415 nm) in 0.2 M Na2HPO4 citric acid (pH 5.0 aqueous buffer containing 1% DMSO) on gradual addition of HSO3− (0–140μM). Each spectrum was recorded 5 min after HSO3− addition. Insert: The relationship between the fluorescence intensity (F415nm) and the bisulfite concentrations. Working conditions: pH 5.0, Reaction time = 5 min.
Fig. 3. Effect of pH on the emission intensity of the probe. Bisulfite concentration: (a) 0 and (b) 100μM.
Fig. 4. Effect of reaction time on the emission intensity of the probe addition reaction system by bisulfite. Bisulfite concentration: (a) blank and (b) 100μM. Detection condition: pH=5.0.
Fig. 5. Emission spectra of the probe (10μM) with various anions (200μM except HSO3- 100μM), the emission spectra was recorded after 5 min. Detection condition: pH=5.0.
Fig. 6.The proposed mechanism for the determination of HSO3−, The 1H-NMR spectra of the DMPCA (top) and DMPCA+10eq. NaHSO3 (bottom) in DMSO-d6.
Fig. 7. (a) MALDI-TOF MS spectrum of DMPCA (DCTB as matrix). (b) MALDI-TOF MS spectrum of DMPCA+HSO3− (DCTB as matrix).
Fig. 8 (a) Fluorescence emission spectra of DMPCA (10μM) in water-polyethylene glycol 400 system with increased viscosity, λex=345 nm. (b) Fluorescence decay curves for DMPCA and DMPCA +HSO3- in water.
Fig. 9. Two-photon excitation spectra of probe DMPCA+HSO3-.
Fig. 10. (a) Bright-field image of HeLa cells incubated with 10μM DMPCA after 30 min of incubation, washed with PBS buffer, λex =700 nm (fluorescence at the blue emission channel); (b) Two-photon image of HeLa cells; (c) The overlay of (a) and (b); (d) Bright-field image of HeLa cells following a 30 min treatment with NaHSO3 (100μM, fluorescence at the blue emission channel); (e) Two-photon image of HeLa cells; (f) The overlay of (d) and (e).
Table Table 1. X-ray crystallography data for DMPCA. Compound
DMPCA
Chemical formula Formula Mass Crystal system Space group a/Å b/Å c/Å α/° β/° γ/° Unit cell volume/Å3 Temperature/K Z No. of reflections measured No. of independent reflections Rint Final R1 values (I > 2σ(I)) Final wR(F2) values (I > 2σ(I)) Final R1 values (all data) Final wR(F2) values (all data) Goodness of fit on F2
C20H15NO3 317.33 Monoclinic P21/c 19.2062(5) 7.0953(2) 12.0369(3) 90 99.979(3) 90 1615.50(8) 291(2) 4 5651 2929 0.0207 0.0459 0.1221 0.0585 0.1350 1.034
S0925-4005(16)30784-5 http://dx.doi.org/doi:10.1016/j.snb.2016.05.099 SNB 20258
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
31-3-2016 17-5-2016 18-5-2016
Please cite this article as: Shenglong Yu, Xiuli Yang, Zhonglong Shao, Yan Feng, Xinguo Xi, Rong Shao, Qingxiang Guo, Xiangming Meng, A TICT based two-photon fluorescent probe for bisulfite anion and its application in living cells, Sensors and Actuators B: Chemical http://dx.doi.org/10.1016/j.snb.2016.05.099 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A TICT based two-photon fluorescent probe for bisulfite anion and its application in living cells Shenglong Yu,a Xiuli Yang,b Zhonglong Shao,a Yan Feng,a Xinguo Xi,b Rong Shao,b Qingxiang Guo, a,c Xiangming Meng,*a,b
a
Department of Chemistry, Anhui University, Hefei, 230601, China.
b
Jiangsu Collaborative
Environmental
Innovation
Protection
Center
Equipments,
for
Ecological
Yancheng
Institute
Building of
Materials
and
Technology, Yancheng
224051, China c
Department of Chemistry, University of Science and Technology of China, Hefei, 230026, China.
*Corresponding author. Fax: +86-551-63861467; Tel: +86-551-63861467 E-mail address: [email protected] (Xiangming Meng).
ABSTRACT A two-photon fluorescent probe (DMPCA) for the detection of bisulfite anion in water was developed based on twisted-intramolecular-charge-transfer (TICT) mechanism. Fluorescence intensity of DMPCA increased dramatically (65 fold) upon the addition of HSO3−. The two-photon absorption cross-section of DMPCA was found to be 725 GM at 700 nm upon the addition of HSO3−. The fluorescent probe showed an excellent selectivity for bisulfite anion over other anions. The nucleophilic addition reaction mechanism with aldehyde was confirmed with NMR and MALDI-TOF. The TICT detection mechanism was confirmed by viscosity-dependent fluorescence spectra and time resolved fluorescence spectra. Moreover, DMPCA was successfully applied to the detection of bisulfite anion in HeLa cells with low cytotoxicity under two-photon excitation.
Keywords: TICT; Two-photon; Fluorescent probe; Coumarin; Bisulfite anion
1. Introduction Bisulphite (HSO3−) is essential preservative for many foods, beverages, and pharmaceutical products to prevent oxidation and bacterial growth and to also control enzymatic reactions during production and storage [1–3]. Moreover, bisulfite has harmful effects on tissue, cells and biomacromolecules. It can cause visible damage such as necrosis, inhibit cell division, induce micronucleus and lead to cell death [4-6]. Therefore, the development of sensitive, selective and low cost methods for the detection of bisulfite is highly needed. Convenient tools, such as optical sensors [7, 8] and chromoreactands [9] have attracted significant research interest during last years. However, fluorescent probes provide the optimal choice to detect biological agents due to its high sensitivity, simplicity for implementation, real-time detection, and good compatibility for bio-samples [10-12]. So far, numbers of small-molecule fluorescent probes for specific detection of bisulfite have been reported [13-21]. However, most of these probes are only applicable in half-water conditions, and they are almost designed on single-photon fluorescence technology. These probes with one-photon microscopy (OPM) require excitation with short-wavelength light (ca. 350–550 nm) that limits their application in deep-tissue imaging, owing to the shallow penetration depth as well as to photo-bleaching, photo-damage, and cellular auto fluorescence. Recently,
two-photon
fluorescence (TPF) probes, which can be excited by two-photon absorption in the NIR wavelength, provided an opportunity to overcome the problems originated from the single-photon fluorescence technology [22-28]. Herein, we report a novel TICT based two-photon fluorescent probe (DMPCA) based on coumarin skeleton (Scheme 1) [29-31]. Coumarin was used as the fluorescent group for their excellent optical properties. N,N-dimethyl group(-N(CH3)2), a typical TICT donor [32-37], was
use as the electron donor. Based on previous results [38-40], phenyl alkynyl was introduced at the 7-position of coumarin to achieve good two-photon fluorescence performance. Aldehydes can selectively react with bisulfite to form aldehyde-hydrogen sulfite adduct [41, 42]. We deem that the aldehyde group of DMPCA can react with bisulfite with high selectivity and cause the change of fluorescence of the probe. Thus, the fluorescent detection signal of bisulfite could be achieved. To the best of our knowledge, DMPCA is the first TICT based two photon fluorescent probe for bisulfite.
Scheme 1. The proposed TICT mechanism for the determination of HSO3−.
2. Experimental section 2.1 General procedures All reagents and solvents were commercially purchased. All reactions were magnetically stirred and monitored by thin layer chromatography (TLC). The 1H NMR (400 MHz) and
13
C NMR (100 MHz) spectra were recorded on a Bruker Avance
spectrometer using DMSO-d6 and CDCl3 as the solvents. Fluorescence spectra were obtained using a HITACHIF-2500 spectrometer. UV-vis absorption spectra were recorded on a Tech-comp UV 1000 spectrophotometer. MS spectra were conducted by Bruker autoflex III MALDI TOF mass spectrometer. The test solution of probe DMPCA (10μM) in 0.2 M Na2HPO4 citric acid (pH 5.0 aqueous buffer containing 1% DMSO) was prepared. The fluorescence quantum yields were detected by HORIB FluoroMax-4P. The two-photon cross section was tested in DMSO with 1mM DMPCA+NaHSO3. TPEF spectra were measured using femtosecond laser pulse and Ti: sapphire system (680–1080 nm, 80 MHz, 140 fs, Chameleon II) as the light source. 2.2 Preparation of the Test Solution
One millimole of inorganic salt (NaHSO3, NaNO3, Na2CO3, NaHSO4, NaF, NaCl, NaCN, KBr, KSCN, KI, Cys·HCl, NaHS and Na2S2O3) was dissolved in distilled water (10 mL) to afford 1×10−1mol/L aqueous solution. The stock solutions were diluted to desired concentrations with water when needed. The resulting solution was shaken well and incubated for 5 min at room temperature before recording the spectra. 2.3 Synthesis of probe DMPCA The DMPCA was easily synthesized via four steps from readily available initial materials with an overall yield of 13.0% (Scheme 2). The structures of DMPCA and intermediates were all confirmed by 1H NMR and 13C NMR.
Scheme 2. Synthetic route of DMPCA.
Compound 1. 3-Iodophenol (10g, 45mmol) was dissolved in anhydrous acetonitrile (160ml), cooled in an ice bath and magnesium chloride (12.8g, 134mmol) added portion-wise over 10 min. Triethylamine (25.3ml, 363mmol) was added to this mixture over 5min, followed by portion-wise addition of paraformaldehyde (5.47g, 636mmol). After complete addition the mixture was heated at reflux for 18.5 hours. The mixture was cooled and quenched by the addition of sat. NH4Cl (350ml) and extracted with EtOAc (3xl50ml). The combined EtOAc layers were washed with sat. NaHCO3 (2x150mml), 1N HCl (2 x 150ml), and sat. NaCl (2x100ml), dried over Na2SO4, filtered and evaporated. The residue was purified by MPLC on silica gel eluting with a gradient rising from 100% hexanes to 20% EtOAc in hexanes. Product containing fractions were combined and evaporated and crystallized from hot hexanes to give of 5.64g the compound 1 (25.5mmol, 50% ). 1H NMR(400 MHz, CDCl3,ppm) : 11.01 (s, 1H),
9.85 (s, 1H), 7.44 (s, 1H), 7.39 (d, J =8.1 Hz, 1H), 7.23 (d, J =8.1 Hz, 1H). 13C NMR (100MHz, CDCl3, ppm): δ 196.12 , 161.31, 134.22 , 129.42 , 127.26 , 119.95 , 105.18. Compound 2. Mixture of 2-hydroxy-4-iodobenzaldehyde (20.31g, 81.89mmol), propionic anhydride (20.99g, 230.5mmol), and K2CO3 (0.5528g, 4mmol) was refluxed until the color of the reaction mixture became brown (16 h). The excess of propionic anhydride was distilled off. The residue was poured into crushed ice, and the pH was adjusted to 7 using NaHCO3. The precipitate was filtered off, washed with water, dried, and recrystallized from a mixture of hexane and ethyl acetate (4:1) to give 12.88g of the compound 2 (45.04mmol, 55%) as white needles. 1H NMR(400 MHz, CDCl3,ppm) :7.64 (s, 1H), 7.55 (d, J=8.1 Hz, 1H), 7.44 (s, 1H), 7.11 (d, J=8.1 Hz, 1H), 2.17 (s, 3H). 13C NMR (100MHz, CDCl3, ppm): δ 161.34 , 153.19, 138.63, 133.52, 127.97, 126.64 , 125.57 , 119.03, 95.44, 17.37. Compound 3. A mixture of the compound 2 (0.89g, 3.1mmol) and SeO2 (0.69 g, 6.2mmol) was ground in a mortar and transferred into a microwave vial, which was sealed and irradiated at 150W and 170oC for 1h. Caution! An extraction funnel was situated over the reaction vial during the reaction with SeO2 to remove noxious vapor. The product was then extracted with EtOAc, concentrated, and purified by flash chromatography to afford 0.48g brown crystals of the compound 3 (1.61mmol, 52%). 1H NMR(400 MHz, CDCl3, ppm) : 10.22 (s, 1H), 8.36 (s, 1H), 7.80 (s, 1H), 7.71 (d, J=8.1 Hz, 1H), 7.37 (d, J=8.2 Hz, 1H). 13C NMR (100MHz, CDCl3, ppm): δ 187.39 , 159.32, 155.15 , 144.90, 134.84 , 131.32 , 126.60 , 122.23, 117.63, 102.05. DMPCA. The mixture of compound 3 (5.43g, 18.1mmol), 4-ethynyl-N,N-dimethylaniline (3.15g, 21.72mmol), PdCl2(PPh3)2 (71.8 mg, 0.905mmol), Et3N (3.66 g, 36.2mmol), and THF (20 mL) was heated at 30oC for 3h. After cooling to room temperature, the mixture was filtered to
remove salts and followed with the addition of 100 mL H2O. The resulting mixture was extracted with EtOAc (50 mL×3). The organic phase was combined and dried with anhydrous Na2SO4, and evaporated to yield a crude product that was recrystallized in EtOAc/PE to give 5.22g of DMPCA (16.48mmol, 91%). 1H NMR (400MHz, CDCl3, ppm) : 10.25 (s, 1H), 8.37 (s, 1H), 7.60 (d, J=8.0 Hz, 1H), 7.43 (t, J=8.7 Hz, 4H), 6.67 (d, J=8.3 Hz, 2H), 3.03 (s, 6H). 13C NMR (100MHz, CDCl3, ppm): δ 187.73, 159.95, 155.44, 150.80, 144.77, 133.27, 131.77, 130.27, 127.93, 120.94, 118.53, 117.03, 111.74, 108.33, 98.30, 87.17, 40.04. MALDI-TOF MS spectrum: [DMPCA-H]- (m/zcalcd =316.11). 2.4 Measurement of Two-photon Absorption Cross-section (ä) Two-photon
excitation
fluorescence
(TPEF)
spectra
were
measured
using
femtosecond laser pulse and Ti: sapphire system (680~1080 nm, 80 MHz, 140 fs, Chameleon II) as the light source. All measurements were carried out in air at room temperature. TPA cross sections were measured using two-photon-induced fluorescence measurement technique. The TPA cross-sections (δ) were determined in acetonitrile with 1mM DMPCA+NaHSO3 by comparing their TPEF according to the following equation:
ref
ref cref nref F c n Fref
Here, the subscripts ref stands for the reference molecule. δ is the TPA cross-section value, c is the concentration of solution, n is the refractive index of the solution, F is the TPEF integral intensities of the solution emitted at the exciting wavelength, and Φ is the fluorescence quantum yield. The δref value of reference was taken from the literature [43]. 2.5 Cell culture and two-photon fluorescence microscopy imaging
For two-photon bio-imaging, HeLa cells were cultured in DMEM supplemented with 10% FCS, penicillin (100μg/mL), and streptomycin (100μg/mL) at 37 oC in a humidified atmosphere with 5% CO 2 and 95% air. Cytotoxicity assays show that DMPCA is safe enough for two-photon bio-imaging at low concentrations, so that the cells were incubated with 10μM DMPCA at 37oC under 5% CO2 for 30 min, washed once and bathed in DMEM containing no FCS prior to imaging and/or HSO3- addition. Then 100μM HSO3was added in the growth medium for 0.5 h at 37 oC, washed 3 times with PBS buffer. Then, cells were imaged on a confocal microscope (Zeiss LSM 510 Meta NLO). Two-photon fluorescence microscopy images of labeled cells were obtained by exciting the probe with a mode-locked titanium-sapphire laser source set at wavelength 700 nm.
3. Results and discussion As shown in Scheme 2, the probe DMPCA was synthesized via four steps from 3-Iodophenol. The structures of DMPCA and the intermediates were all confirmed by 1H-NMR and
13
C-NMR. The structure of DMPCA was also confirmed by X-ray crystallography analysis
(CCDC 1401985). The single crystals of DMPCA grew from anhydrous ether–petroleum ether (60–90 °C) solution. The crystal structure of DMPCA was depicted in Fig. 1. The parameters associated with the crystal data are shown in Table 1.
Fig. 1. Molecular structure of DMPCA (ORTEP drawings of DMPCA showing thermal ellipsoids at 30% probability level).
Table 1. X-ray crystallography data for DMPCA.
3.1 UV–vis and fluorescence spectra study Fig. 2 shows the change of the fluorescence spectra of DMPCA (10uM in 0.2M citric acid buffer, pH=5.0, λex =345 nm) upon the addition of HSO3−. Upon the increasing of HSO3− concentration, the initial fluorescence intensity at 415 nm increased linearly with 65 fold and reached a plateau when 10 equiv. of HSO3− was added, as show in Fig. S1, and the limit of detection (LOD) was calculated to be 1.38 10-7μM (σ=0.093, k=2.02, R2=0.994). In addition, Fig. S2 shows the change on the UV-vis spectrum when HSO3− was added to the Na2HPO4 citric acid buffer (0.2 M, pH 5.0) solution containing probe DMPCA (10μM). Upon the addition of HSO3−, the absorption peaks of DMPCA at 345 nm was increased clearly, which may indicate the formation of new compound.
Fig. 2. Emission spectral changes for the probe DMPCA (10μM) (λex= 345 nm, λem = 415 nm) in 0.2 M Na2HPO4 citric acid (pH 5.0 aqueous buffer containing 1% DMSO) on gradual addition of HSO3− (0–140μM). Each spectrum was recorded 5 min after HSO3− addition. Insert: The relationship between the fluorescence intensity (F415nm) and the bisulfite concentrations. Working conditions: pH 5.0, Reaction time = 5 min.
3.2 The pH stability The effect of pH on the fluorescence properties of the system was investigated (Fig. 3). It is obvious that the fluorescent signal of the probe is stable within pH scale 3.2-9.3. However, the fluorescence signal of the probe with bisulfite anion increased from pH 3.2 to 5.0, and decreased at pH values above 6.0. The aldehyde-bisulfite adduct is stable within the pH 5.0 to 6.0. These result is similar to previous reported bisulfite probes [44], thus, the pH 5.0 buffer solution was selected as experimental detection condition.
Fig. 3. pH effect on the emission intensity of DMPCA. Bisulfite concentration: (a) 0 and (b) 100μM.
3.3. Response time The response time of DMPCA to bisfute was also studied in Na2HPO4/citric acid buffer (0.2 M, pH 5.0) solution. As shown in Fig. 4, we can see that the addition reaction between DMPCA and bisfulite can be finished within 2 minutes which is very rapid compared with other bisulfite probes [45, 46]. The fast response time makes DMPCA applicable in real detection of bisulfite.
Fig. 4. The response time of DMPCA(10uM) to bisulfite. Bisulfite concentration: (a) blank and (b) 100μM, detection condition: pH=5.0.
3.4 Selectivity over other anions Ion selectivity study was performed in Na2HPO4 citric acid buffer (0.2 M, pH 5.0) solution. The optical responses of DMPCA (10μM) to various anions were investigated by the emission spectroscopy. As shown in Fig. 5, none of the anions except HSO3− induced the change of the fluorescence. The color change of fluorescence DMPCA to various ions was shown in Fig. S3, it’s obvious that only HSO3− turn on the blue fluorescence of the solution of DMPCA. Competitive experiments were also carried out using HSO3− (100μM) mixed with other anions (200μM). As shown in Fig. S4, other anions present little impacts on the detecting signal (I415nm) of DMPCA. These results suggest that DMPCA has good selectivity for HSO3− over other anions. Fig. 5. Emission spectra of the probe (10μM) with various anions (200μM except HSO3- 100μM), the emission spectra was recorded after 5 min. Detection condition: pH=5.0.
3.5. Nucleophilic addition mechanism To analyze the reaction mechanism, H-NMR and MS titration was carried out. Form 1H NMR spectrum, we can see that the aldehyde proton at around δ 10.25 was dramatically shifted to 4.79 (Fig. 6). These results indicated that the aldehyde group were reacted with bisulfite ions under detection condition. The reaction mixture of DMPCA with NaHSO3 was characterized by MALDI-TOF MS spectrum. As shown in Fig. 7, DMPCA revealed a main peak at 315.88 before the addition of NaHSO3, corresponding to the species [DMPCA-H]- (m/zcalcd =316.11). After the addition of NaHSO3, a relatively weak peak at about 420.13 appeared coinciding exactly with that for the adduct species [DMPCA+NaHSO3-H]- (m/zcalcd = 420.06). The above data suggested that the nucleophilic addition reaction of the probe’s formyl group and the bisulfate ion was the origin of the detection signal. .
Fig. 6.The proposed mechanism for the detection of HSO3−, The 1H-NMR spectra of the DMPCA (top) and DMPCA+10eq. NaHSO3 (bottom) in DMSO-d6.
Fig. 7. (a) MALDI-TOF MS spectrum of DMPCA (DCTB as matrix). (b) MALDI-TOF MS spectrum of DMPCA+ NaHSO3 (DCTB as matrix).
3.6. The viscosity-dependent fluorescence spectra and fluorescence lifetime To further understand the TICT mechanism of DMPCA, the viscosity-dependent fluorescence emission was carried out in the water-polyethylene glycol system. As shown in Fig. 8(a), with the increase of the solvent viscosity, the significant fluorescence enhancement (about
30-folds) was observed. Increased solvent viscosity would prohibit the rotation of the C-C bond of DMPCA (as shown in scheme 1), thereby prevent the non-radiative energy loss of the excited singlet state. As a result, the fluorescence emission was increased subsequently. On the other hand, fluorescence lifetime of DMPCA with and without bisulfate ion was detected by time resolved fluorescent spectra. As shown in Fig. 8(b) and Table S1, the fluorescence decay curve of DMPCA was biexponential (τ1=0.65 ns, τ2=2.74 ns), in which the slow component (τ2=2.74 ns, 33%) was attributed to the TICT state. However, upon addition of bisulfate ion the fluorescence decay curve of DMPCA was changed into monoexponential (τ=1.08 ns) due to the disappearance of TICT state. Thereby, the fluorescence emission was increased after HSO3- addition. These results indicated that the change of spectra of DMPCA was attributed to TICT process.
Fig. 8 (a) Fluorescence emission spectra of DMPCA (10μM) in water-polyethylene glycol 400 system with increased viscosity, λex=345 nm. (b) Fluorescence decay curves for DMPCA and DMPCA +HSO3- in water.
3.7. Two-photon absorption cross-sections The two-photon absorption cross-sections of DMPCA+HSO3- were detected using the two-photon induced fluorescence measurement spectra. As shown in Fig. 9, the maximum two-photon absorption cross-section (δmax) value of DMPCA+HSO3- is 725 GM at 700 nm (Φ=0.07). The large two-photon absorption cross-sections of DMPCA+HSO3- suggested that probe DMPCA would be a good two-photon probe for bisulfite ion.
Fig. 9. Two-photon cross-sections of DMPCA+HSO3-.
3.8 Cell cytotoxicity and fluorescence microscopy imaging Cell cytotoxicity assays were conducted using HeLa cells to test the cytotoxicity of DMPCA. The cell viability remains more than 95% after treated with 10μM DMPCA for 24 h (Fig. S5). The low cytotoxicity of DMPCA confirmed that the probe is safe for bio-imaging. With the above data in hand, we finally investigated the utility of DMPCA to image HSO3− in living cells under one-photon and two-photon excitation. HeLa cells were cultured and stained with DMPCA (10μM) within 30 min and washed by PBS buffer and then treated with HSO3− (100μM) for another 30 min. TPM images were obtained by exciting the probe at wavelength 700 nm. The blue emission channel was chosen as the detecting windows for DMPCA and DMPCA+HSO3−. As shown in Fig. 10, before the addition of HSO3−, the blue emission channel exhibited no fluorescence. After HSO3− addition, the fluorescence of the blue emission channel exhibited strong fluorescence dramatically. This indicated that DMPCA is cell permeable. The fluorescence images generated from the blue emission channel demonstrated that DMPCA can offer a simple and visible way to detect HSO3- in vivo in real time under two-photon excitation.
Fig. 10. (a) Bright-field image of HeLa cells incubated with 10μM DMPCA after 30 min of incubation, washed with PBS buffer, λex =700 nm (fluorescence at the blue emission channel); (b) Two-photon image of HeLa cells; (c) The overlay of (a) and (b); (d) Bright-field image of HeLa cells following a 30 min treatment with NaHSO3 (100μM, fluorescence at the blue emission channel); (e) Two-photon image of HeLa cells; (f) The overlay of (d) and (e).
4. Conclusions
In conclusion, we developed a TICT based two-photon fluorescent probe (DMPCA) for the detection of bisulfite anion in water. Upon the addition of bisulfite anion, the probe displayed obvious fluorescence enhancement (about 65 folds). Taking advantage of the nucleophilic addition reaction with aldehyde, the probe has exhibited a sensitive and selective response to the bisulfite anion against other anions. DMPCA shows maximum two-photon absorption cross-sections (725 GM) at 700nm upon the addition of bisulfite anion. Furthermore, the probe was successfully applied to the detection of the bisulfite anion in living cells under two-photon excitation.
Acknowledgements This work was supported by National Natural Science Foundation of China (21102002, 21272223, 21372005), The research fund of Jiangsu Collaborative Innovation Center for Ecological Building Materials and Environmental Protection Equipments and Anhui University.
Biographies Shenglong Yu is studying for master degree in Anhui University. He received his bachelor of science in chemistry from the department of chemistry at Anhui University.
XiuLi Yang
is an assitant professor at Jiangsu Collaborative
Innovation Center
for Ecological Building Materials and Environmental Protection Equipments and Yancheng Institute of Technology. Her research focus on the design and synthesis of functional Metal-Organic
Frameworks for sensing application.
Zhonglong Shao is an associate professor in the department of chemistry at Anhui University. His current research interests are chemosensors and theoretical calculation.
Yan Feng is an associate professor in the department of chemistry at Anhui University. Her current research interests are chemosensors and organometallic chemistry.
Xinguo
Xi
is
a
professor
at
Ecological Building Materials and Insti-tute
of
Technology.
His
Jiangsu
Collaborative
Environmental research
focus
Innovation
Protection Equipments on
the
design
Center and
and
for
Yancheng
synthesis
of
functional Metal-Organic Frameworks for sensing and catalysis application.
Rong Shao
is a
professor
at
Ecological Building Materials and Institute
Jiangsu
Collaborative
Environmental
Innovation
Protection Equipments
Center and
of
for
Yancheng Technology.
His research interest lies in chemical environmental protection and bio-chemical.
Qiangxiang Guo is a professor in department of chemistry at University of Science and technology of China and joined the department of chemistry at Anhui University as a distinguished professor, in 2015. His research interest is physical organic chemistry.
Xiangming Meng is an associate professor in the department of chemistry at Anhui University. His current research interests are chemosensors and nanoclusters.
References [1] H. Tian, J. Qian, Q. Sun, H. Bai, W. Zhang, Colorimetric and ratiometric fluorescent detection of sulfite in water via cationic surfactant-promoted addition of sulfite to α, β-unsaturated ketone, Analytica chimica acta 788 (2013) 165-170. [2] Y. Sun, S. Fan, S. Zhang, D. Zhao, L. Duan, R. Li, A fluorescent turn-on probe based on benzo [e] indolium for bisulfite through 1,4-addition reaction, Sensors and Actuators B: Chemical 193 (2014) 173-177. [3] G. Xu, H. Wu, X. Liu, R. Feng, Z. Liu, A simple pyrene-pyridinium-based fluorescent probe for colorimetric and ratiometric sensing of sulfite, Dyes and Pigments 120 (2015) 322-327. [4] W. Xu, C.L. Teoh, J. Peng, D. Su, L. Yuan, Y.T. Chang, A mitochondria-targeted ratiometric fluorescent probe to monitor endogenously generated sulfur dioxide derivatives in living cells, Biomaterials 56 (2015) 1-9. [5] J. Xu, J. Pan, X. Jiang, C. Qin, L. Zeng, H. Zhang, J.F. Zhang, A mitochondria-targeted ratiometric fluorescent probe for rapid, sensitive and specific detection of biological SO2 derivatives in living cells, Biosensors and Bioelectronics 77 (2016) 725-732. [6] X. Cheng, H. Jia, J. Feng, J. Qin, Z. Li, “Reactive” probe for hydrogen sulfite: Good ratiometric response and bioimaging application, Sensors and Actuators B: Chemical 184 (2013) 274-280. [7] S.S.M. Hassan, M.S.A. Hamza, A.H.K. Mohamed, A novel spectrophotometric method for batch and flow injection determination of sulfite in beverages, Analytica Chimica Acta 570 (2006) 232–239; [8] P.D. Tzanavaras, E. Thiakouli, D.G. Themelis, Hybrid sequential injectionflow injection manifold for the spectrophotometric determination of total sulfite in wines using o-phthalaldehyde and gas-diffusion, Talanta 77 (2009) 1614–1619. [9] G.J. Mohr, A chromoreactand for the selective detection of HSO3− based on the reversible bisulfite addition reaction in polymer membranes, Chemical Communications (2002) 2646–2647. [10] C. Huang, T. Jia, M.F. Tang, Q. Yin, W.P. Zhu, Y.F. Xu, X.H. Qian, et al. Selective and Ratiometric Fluorescent Trapping and Quantification of Protein Vicinal Dithiols and in Situ Dynamic Tracing in Living Cells, Journal of the American Chemical Society 136 (2014)
14237–14244. [11] Y.C. Chen, C.C. Zhu, Z.H. Yang, J.J. Chen, Y.F. He, J.J. Cen, Z.J. Guo, A ratiometric fluorescent probe for rapid detection of hydrogen sulfide in mitochondria, Angewandte Chemie 125 (2013) 1732–1735. [12] A.T. Wrobel, T.C. Johnstone, A. Deliz Liang, S.J. Lippard, P. Rivera-Fuentes, A fast and selective near-infrared fluorescent sensor for multicolor imaging of biological nitroxyl (HNO), Journal of the American Chemical Society 136 (2014) 4697-4705. [13] M.G. Choi, J. Hwang, S. Eor, S.K. Chang, Chromogenic and fluorogenic signaling of sulfite by selective deprotection of resorufin levulinate, Organic letters 12 (2010) 5624-5627. [14] G. Li, Y. Chen, J. Wang, Q. Lin, J. Zhao, L. Ji, H. Chao, A dinuclear iridium (III) complex as a visual specific phosphorescent probe for endogenous sulphite and bisulphite in living cells, Chemical Science 4 (2013) 4426-4433. [15] J. Chao, Y. Zhang, H., Wang, Y. Zhang, F. Huo, C. Yin, Y. Wang, Fluorescent Red GK as a fluorescent probe for selective detection of bisulfite anions, Sensors and Actuators B: Chemical 188 (2013) 200-206. [16] Y.Q. Sun, J. Liu, J. Zhang, T. Yang, W. Guo, Fluorescent probe for biological gas SO2 derivatives bisulfite and sulfite, Chemical Communications 49 (2013) 2637-2639. [17] H. Tian, J. Qian, Q. Sun, C. Jiang, R. Zhang, W. Zhang, A coumarin-based fluorescent probe for differential identification of sulfide and sulfite in CTAB micelle solution, Analyst 139 (2014) 3373-3377. [18] M.Y. Wu, K. Li, C.Y. Li, J.T. Hou, X.Q. Yu, A water-soluble near-infrared probe for colorimetric and ratiometric sensing of SO2 derivatives in living cells, Chemical Communications 50 (2014) 183-185. [19] L. Zhu, J. Xu, Z. Sun, B. Fu, C. Qin, L. Zeng, X. Hu, A twisted intramolecular charge transfer probe for rapid and specific detection of trace biological SO2 derivatives and bio-imaging applications, Chemical Communications 51 (2015) 1154-1156. [20] X. Dai, T. Zhang, Z.F. Du, X.J. Cao, M.Y. Chen, S.W. Hu, ... & B.X. Zhao, An effective colorimetric and ratiometric fluorescent probe for bisulfite in aqueous solution, Analytica chimica acta 888 (2015) 138-145.
[21] D.P. Li, Z.Y. Wang, X.J. Cao, J. Cui, X. Wang, H.Z. Cui, ... & B.X. Zhao, A mitochondria-targeted fluorescent probe for ratiometric detection of endogenous sulfur dioxide derivatives in cancer cells, Chemical Communications 52 (2016) 2760--2763. [22] Q. Xu, C.H. Heo, G. Kim, H.W. Lee, H.M. Kim, J. Yoon, Development of Imidazoline-2-Thiones Based Two-Photon Fluorescence Probes for Imaging Hypochlorite Generation in a Co-Culture System, Angewandte Chemie 127 (2015) 4972-4976. [23] S.K. Bae, C.H. Heo, D.J. Choi, D. Sen, E. H. Joe, B.R. Cho, H.M. Kim, A ratiometric two-photon fluorescent probe reveals reduction in mitochondrial H2S production in Parkinson’s disease gene knockout astrocytes, Journal of the American Chemical Society 135 (2013) 9915-9923. [24] H. Zhang, J.L. Fan, J.Y. Wang, S.Z. Zhang, B.R. Dou, X.J. Peng, An Off–On COX-2-Specific Fluorescent Probe: Targeting the Golgi Apparatus of Cancer Cells, Journal of the American Chemical Society 135 (2013) 11663-11669. [25] F. Miao, W.J. Zhang, Y.M. Sun, R.Y. Zhang, Y. Liu, F.Q. Guo, X.Q. Yu, Novel fluorescent probes for highly selective two-photon imaging of mitochondria in living cells, Biosensors and Bioelectronics 55 (2014) 423-429. [26] L.Y. Zeng, S.Y. Chen, T. Xia, C. Zhong, Z.H. Liu, Designing two-photon fluorescent probes based on the target-induced enhancement of the absorption cross-section, Chemical Communications 50 (2014) 11139-11142. [27] L.Y. Zhou, X.B. Zhang, Q.Q. Wang, Y.F. Lv, G.J. Mao, A.L. Luo, Y.X. Wu, Y. Wu, J. Zhang, W.H. Tan, Molecular Engineering of a TBET-Based Two-Photon Fluorescent Probe for Ratiometric Imaging of Living Cells and Tissues, Journal of the American Chemical Society 136 (2014) 9838-9841. [28] L. Yuan, W.Y. Lin, H. Chen, S.S. Zhu, L.W. He, A Unique Family of Rigid Analogues of the GFP Chromophore with Tunable Two-Photon Action Cross-Sections for Biological Imaging, Angewandte Chemie International Edition 52 (2013) 10018-10022. [29] B. Chen, Y. Ding, X. Li, W. Zhu, J. P. Hill, K. Ariga, & Y. Xie, Steric hindrance-enforced distortion as a general strategy for the design of fluorescence “turn-on” cyanide probes, Chemical Communications 49 (2013) 10136-10138.
[30] Y. Xie, Y. Ding, X. Li, C. Wang, J.P. Hill, K. Ariga, ... & W. Zhu, Selective, sensitive and reversible
“turn-on”
fluorescent
2′-dipyridylaminoanthracene–Cu2+
cyanide
ensembles, Chemical
probes
based
Communications
on 48
2, (2012)
11513-11515. [31] Y. Ding, Y. Tang, W. Zhu, & Y. Xie, Fluorescent and colorimetric ion probes based on conjugated oligopyrroles, Chemical Society Reviews 44 (2015) 1101-1112. [32] Y. Ding, W.H. Zhu, & Y. Xie, Development of Ion Chemosensors Based on Porphyrin Analogues, Chemical Reviews (2016). [33] S. I. Reja, I. A. Khan, V. Bhalla, M. Kumar, A TICT based NIR-fluorescent probe for human serum albumin: a pre-clinical diagnosis in blood serum, Chemical Communications 52 (2016) 1182-1185. [34] V. Tharmaraj, K. Pitchumani, An acyclic, dansyl based colorimetric and fluorescent chemosensor for Hg (II) via twisted intramolecular charge transfer (TICT), Analytica Chimica Acta 751 (2012) 171–175. [35] J. S. Yang, C. K. Lin, A. M. Lahoti, C. K. Tseng, Y. H. Liu, G. H. Lee, S. M. Peng, Effect of Ground-State Twisting on the trans→ cis Photoisomerization and TICT State Formation of Aminostilbenes , the Journal of Physical Chemistry A 113 (2009) 4868–4877. [36] M. Cigáň, J. Donovalová, V. Szöcs, J. Gašpar, K. Jakusová, A. Gáplovský, 7-(Dimethylamino) coumarin-3-carbaldehyde and its phenylsemicarbazone: TICT excited state modulation, fluorescent H-aggregates, and preferential salvation, the Journal of Physical Chemistry A 117 (2013) 4870−4883. [37] X.J. Wei, X.L. Yang, Y. Feng, P. Ning, H.Z. Yu, M.Z. Zhu, X.G. Xi, Q.X. Guo, X.M. Meng, A TICT based two-photon fluorescent probe for cysteine and homocysteine in living cells, Sensors and Actuators B: Chemical 231 (2016) 285−292. [38] H.J. Yin, B.C. Zhang, H.Z. Yu, L. Zhu, Y. Feng, M.Z. Zhu, X.M. Meng, Two-Photon Fluorescent Probes for Biological Mg2+ Detection Based on 7-Substituted Coumarin, The Journal of organic chemistry 80 (2015) 4306-4312. [39] X.M. Meng, S.X. Wang, Y.M. Li, M.Z. Zhu, Q.X. Guo, 6-Substituted quinoline-based ratiometric two-photon fluorescent probes for biological Zn2+ detection, Chemical Communications 48 (2012) 4196-4198.
[40] L. Yuan, W. Lin, Z. Cao, J. Wang, B. Chen, Development of FRET-Based Dual-Excitation Ratiometric Fluorescent pH Probes and Their Photocaged Derivatives, Chemistry-A European Journal 18 (2012) 1247-1255. [41] L.C. de Azevedo, M.M. Reis, L.F. Motta, G.O.D. Rocha, L.A. Silva, J.B. de Andrade, Evaluation of the formation and stability of hydroxyalkyl sulfonic acids in wines, Journal of agricultural and food chemistry 55 (2007) 8670-8680. [42] D.P. Kjell, B.J. Slattery, M.J. Semo, A novel, nonaqueous method for regeneration of aldehydes from bisulfite adducts, The Journal of organic chemistry 64 (1999) 5722-5724. [43] O. Varnavski, T. Goodson, L. Sukhomlinova, R. Twieg, Ultrafast Exciton Dynamics in a Branched Molecule Investigated by Time-Resolved Fluorescence, Transient Absorption, and Three-Pulse Photon Echo Peak Shift Measurements, Journal of Physical Chemistry B 108 (2004) 10484-10492. [44] Y. Yang, F. Huo, J. Zhang, Z. Xie, J. Chao, C. Yin, X. Yan, A novel coumarin-based fluorescent probe for selective detection of bissulfite anions in water and sugar samples, Sensors and Actuators B: Chemical 166 (2012) 665-670. [45] C. Wang, S. Feng, L. Wu, S. Yan, C. Zhong, P. Guo, X. Zhou, A new fluorescent turn-on probe for highly sensitive and selective detection of sulfite and bisulfite, Sensors and Actuators B: Chemical 190 (2014) 792-799. [46] W. Chen, Q. Fang, D. Yang, H. Zhang, X. Song, J. Foley, Selective, Highly Sensitive Fluorescent Probe for the Detection of Sulfur Dioxide Derivatives in Aqueous and Biological Environments, Analytical chemistry 87 (2014) 609-616.
Figures and schemes
Scheme 1. The proposed TICT mechanism for the determination of HSO3−.
Scheme 2. Synthetic route of DMPCA.
Fig. 1. Molecular structure of DMPCA (ORTEP drawings of DMPCA showing thermal ellipsoids at 30% probability level).
Fig. 2. Emission spectral changes for the probe DMPCA (10μM) (λex= 345 nm, λem = 415 nm) in 0.2 M Na2HPO4 citric acid (pH 5.0 aqueous buffer containing 1% DMSO) on gradual addition of HSO3− (0–140μM). Each spectrum was recorded 5 min after HSO3− addition. Insert: The relationship between the fluorescence intensity (F415nm) and the bisulfite concentrations. Working conditions: pH 5.0, Reaction time = 5 min.
Fig. 3. Effect of pH on the emission intensity of the probe. Bisulfite concentration: (a) 0 and (b) 100μM.
Fig. 4. Effect of reaction time on the emission intensity of the probe addition reaction system by bisulfite. Bisulfite concentration: (a) blank and (b) 100μM. Detection condition: pH=5.0.
Fig. 5. Emission spectra of the probe (10μM) with various anions (200μM except HSO3- 100μM), the emission spectra was recorded after 5 min. Detection condition: pH=5.0.
Fig. 6.The proposed mechanism for the determination of HSO3−, The 1H-NMR spectra of the DMPCA (top) and DMPCA+10eq. NaHSO3 (bottom) in DMSO-d6.
Fig. 7. (a) MALDI-TOF MS spectrum of DMPCA (DCTB as matrix). (b) MALDI-TOF MS spectrum of DMPCA+HSO3− (DCTB as matrix).
Fig. 8 (a) Fluorescence emission spectra of DMPCA (10μM) in water-polyethylene glycol 400 system with increased viscosity, λex=345 nm. (b) Fluorescence decay curves for DMPCA and DMPCA +HSO3- in water.
Fig. 9. Two-photon excitation spectra of probe DMPCA+HSO3-.
Fig. 10. (a) Bright-field image of HeLa cells incubated with 10μM DMPCA after 30 min of incubation, washed with PBS buffer, λex =700 nm (fluorescence at the blue emission channel); (b) Two-photon image of HeLa cells; (c) The overlay of (a) and (b); (d) Bright-field image of HeLa cells following a 30 min treatment with NaHSO3 (100μM, fluorescence at the blue emission channel); (e) Two-photon image of HeLa cells; (f) The overlay of (d) and (e).
Table Table 1. X-ray crystallography data for DMPCA. Compound
DMPCA
Chemical formula Formula Mass Crystal system Space group a/Å b/Å c/Å α/° β/° γ/° Unit cell volume/Å3 Temperature/K Z No. of reflections measured No. of independent reflections Rint Final R1 values (I > 2σ(I)) Final wR(F2) values (I > 2σ(I)) Final R1 values (all data) Final wR(F2) values (all data) Goodness of fit on F2
C20H15NO3 317.33 Monoclinic P21/c 19.2062(5) 7.0953(2) 12.0369(3) 90 99.979(3) 90 1615.50(8) 291(2) 4 5651 2929 0.0207 0.0459 0.1221 0.0585 0.1350 1.034