Cite this: RSC Advances, 2013, 3,14543
A polymer-based turn-on fluorescent sensor for specificdetection of hydrogen sulfide3
Received 1st March 2013,Accepted 15th May 2013
DOI: 10.1039/c3ra41019f
www.rsc.org/advances
Kai Sun, Xiaoli Liu, Yanyun Wang and Zhaoqiang Wu*
Hydrogen sulfide (H2S) is accepted as a third ‘‘gasotransmitter’’ of human physiology and pathology but
remains difficult to study, in large part because of the lack of methods for the selective monitoring of this
small signaling molecule in live biological specimens. We now report a new reaction-based polymeric
fluorescent sensor for selective imaging of H2S in living cells. A novel functional monomer, 2-allyl-1,3-
dioxo-2,3-dihydro-1H-benzo[de]isoquinoline-6-sulfonyl azide (AISA) was firstly synthesized and copoly-
merized with styrene to obtain a polymeric fluorescent sensor material. AISA and poly(styrene-co-AISA)
(PSAISA) showed a fast turn-on fluorescence signal enhancement and a high selectivity for hydrogen
sulfide (H2S) over other biologically relevant species including HSO32, SO4
22, S2O322 and cysteine.
Furthermore, upon reaction with H2S, PSAISA gave a strong spectral response changing from colorless to
bright yellow. FT-IR and 1H NMR data confirmed that the fluorescence enhancement of PSAISA was caused
by the reduction of sulfonyl azide to sulfonamide in the presence of H2S. This property was successfully
used to image H2S in living cells, thus demonstrating the potential of this material in biosensor
applications.
1. Introduction
Fluorescence detection methods are not only simple but highlysensitive and selective, and allow in situ real-time measure-ments. Fluorescent chemosensors have therefore attractedincreasing attention.1–3 Compared with small molecule fluor-escent sensors, polymer-based fluorescent sensors havepotential advantages in stability, reusability, automatic signaldetection and the fabrication of devices.4 In recent yearspolymer-based fluorescent sensors have been developed fordetecting anions,5 cations6 and molecules,7 and for measuringpH8 and temperature.9
Recent studies show that hydrogen sulfide can be generatedin mammalian tissue mainly by the action of the enzymescystathionine b-synthase (CBS) and cystathionine c-lyase (CSE)on L-cysteine.10 Endogenous H2S affects several physiologicaland pathological processes, including blood vessel relaxation,arterial contraction, neurotransmission, regulation of inflam-mation, cardioprotection, neuroprotection, neurotoxicity, andinsulin release.11 Indeed H2S is accepted as a third ‘‘gaso-
transmitter’’ after nitric oxide (NO) and carbon monoxide(CO).12 Thus it is of vital importance to establish new methodswith good selectivity and sensitivity for the detection of H2Sdue to its role in various physiological processes as well as itsinherent toxicity.13
Colorimetric,14,15 electrochemical16,17 and gas chromato-graphy18,19 assays have been used in the detection of H2S.However, these methods do not allow real-time monitoring ofH2S in tissues, organs and blood. Furthermore, tedious samplepreparation processes and harsh experimental conditionslimit the development of these methods.13 Compared withthese traditional analytical methods, fluorescent H2S sensorshave seen rapid development in recent years. These sensorsare of several types based on: (1) the reduction of RN3 to theparent amine moiety,20–29 (2) two step nucleophilic reac-tions,30–33 (3) intramolecular charge transfer,34–36 (4) Michael-addition–cyclization,37 (5) excited state intramolecular protontransfer (ESIPT),38,39 (6) thiolysis of dinitrophenyl ether,40 (7)two-photon probe41 and (8) fluorescent proteins.42 However,all of the fluorescent H2S sensors so far reported are based onsmall molecules. The design of fluorescent H2S sensors for usein continuous online systems is still a challenge because of thelimitations of small molecules in the fabrication of devices.Some of these limitations could be overcome by the use ofpolymeric sensors, but to the best of our knowledge nopolymeric fluorescent sensor has been reported for thedetection of H2S.
Herein, we report, for the first time, a new kind ofpolymeric fluorescent sensor polystyrene-co-poly(2-allyl-1,3-
Jiangsu Key Laboratory of Advanced Functional Polymer Design and Application,
Department of Polymer Science and Engineering, College of Chemistry, Chemical
Engineering and Materials Science, Soochow University, 199 Ren’ai Road, Suzhou,
215123, P. R. China. E-mail: [email protected]; Fax: +86-512-65880583;
Tel: +86-512-65884279
3 Electronic supplementary information (ESI) available: 1H NMR spectra ofcompound 2 and AISA, FT-IR spectra of compound 2 and AISA, TEM images ofPSAISA, fluorescence spectra of PSAISA in the presence of different biologicallyrelevant anions. See DOI: 10.1039/c3ra41019f
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dioxo-2,3-dihydro-1H-benzo[de]isoquinoline-6-sulfonyl azide)(PSAISA) for H2S detection. The AISA residues provide thefluorescent signal. The data from the fluorescence spectra andintracellular fluorescence images suggest that PSAISA is asensitive and selective H2S detector. Thus PSAISA may beuseful for the measurement of H2S in medical diagnostic,environmental and industrial applications.
2. Materials and methods
2.1. Materials
4-Sulfo-1,8-naphthalic anhydride potassium salt was pur-chased from Sigma-Aldrich Chemicals and used as received.Sodium azide, ammonium persulfate (APS), sodium docecylsulfate (SDS), styrene, allylamine and thionyl chloride werepurchased from Sinopharm Chemical Reagent Co., Ltd.Thionyl chloride and styrene were purified according tostandard procedures. Deionized water purified using aMillipore water purification system to give a minimumresistivity of 18.2 MV cm was used in all experiments. Allsolvents were of analytical grade and used as received. TheChina Center of Type Culture Collection (CCTCC) supplied theHeLa cells.
2.2. Instruments and measurements1H NMR spectra were acquired on a Varian mercury-400spectrometer, using tetramethylsilane (TMS) as an internalstandard. Chemical shifts are reported in parts per million (d)and are referenced to residual protic solvent resonances. Thefollowing abbreviations are used in describing NMR couplings:(s) singlet, (d) doublet, (t) triplet and (b) broad. IR spectra weremeasured on a Nicolet 6700 FT-IR spectrometer as KBr pellets(Thermo Scientific, USA). UV-visible spectra were acquiredusing a HITACHI U-3900 spectrophotometer (Tokyo, Japan).Fluorescence spectra were obtained on a Fluoromax-4 spectro-fluorimeter (HORIBA JobinYvon, USA). Transmission electro-nic microscopy (TEM) images were obtained using a Tecnai G2F20 S-Twin microscope (FEI, USA).
The quantum yields of fluorescence (WF) of the AISA probe(10 mM in ethanol) were calculated relatively to rhodamine B(Wref = 0.65 in ethanol)43 as a reference compound according toeqn (1), where Aref, Sref, nref and Asample, Ssample, nsample
represent the absorbance at the exited wavelength, theintegrated emission band area and the solvent refractive indexof the standard and the sample, respectively.
WF~WrefSsample
Sref
� �Aref
Asample
� �nsample
2
nref2
� �(1)
The detection limit was calculated based on the fluores-cence titration. The fluorescence emission spectrum of theAISA probe was measured five times and the standarddeviation of five replicate measurements of the AISA probewas achieved. To obtain the slope, the fluorescence intensity at530 nm was plotted against the concentration of Na2S. Thedetection limit was calculated according to equation (2):
Detection limit = 3s/k (2)
where s is the standard deviation of the blank measurement, kis the slope of fluorescence intensity vs. Na2S concentration.
2.3. The synthesis of the functional monomer
2.3.1. 1,3-Dioxo-1,3-dihydrobenzo[de]isochromene-6-sulfo-nyl chloride (3). 4-Sulfo-1,8-naphthalic anhydride potassiumsalt (1.6 g, 5.0 mmol) was dissolved in a mixture of 30 mLthionyl chloride and a few drops of N,N-dimethylformamide.The reaction mixture was refluxed in the dark for 12 h, allowedto cool to room temperature and then added slowly to an ice–water mixture to give the desired product as a yellowishpowder (1.4 g, 94% yield). The crude product was used directlywithout purification.
2.3.2. 1,3-Dioxo-1,3-dihydrobenzo[de]isochromene-6-sulfo-nyl azide (2). 1,3-Dioxo-1,3-dihydrobenzo[de]isochromene-6-sulfonyl chloride (1.4 g, 4.7 mmol) and sodium azide (0.33 g,5.0 mmol) were added to 50 mL acetone. The reactant wasrefluxed in the dark for 12 h. The reaction mixture was allowedto cool to room temperature and was added slowly to an ice–water mixture to give the desired product as a yellowishpowder (1.21 g, 81% yield). 1H NMR (400 MHz, DMSO-d6), d
(ppm): 9.28 (d, 1H, ArH), 8.49 and 8.45 (each d, 1H, ArH), 8.20(d, 1H, ArH), 7.91(t, 1H, ArH). IR (KBr, cm21): 2130 (s, v(N3)),1766, 1741 (s, v(CO–O–CO)) (ESI,3 Fig. S1 and S3).
2.3.3. 2-Allyl-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinoline-6-sulfonyl azide (AISA). 1,3-Dioxo-1,3-dihydrobenzo[de]isochromene-6-sulfonyl azide (0.6 g, 2.0 mmol) and allylamine (0.2 mL, 4.6 mmol)were added to 30 mL ethanol. The reaction mixture was refluxed inthe dark for 12 h. After that, the reaction mixture was cooled to 0 uCto precipitate the product, which was isolated by filtration,recrystallized from ethanol, and dried under vacuum (0.52 g, 73%yield). 1H NMR (400 MHz, CDCl3), d (ppm): 8.68 (d, 1H, ArH), 8.61 (d,1H, ArH), 8.47 (d, 1H, ArH), 7.78 (t, 1H, ArH), 7.49 (d, 1H, ArH), 5.99(m, 1H,LCH), 5.35 and 5.21 (each d, 1H, CH2L), 4.72 (d, 2H, CH2). IR(KBr, cm21): 2130 (s, v(N3)), 1698 (s, v(CHLCH2)), 1658 (s, v(CO–N–CO)) (ESI,3 Fig. S2 and S3).
2.4. The preparation of PSAISA
The polymeric fluorescent sensor materials were obtained byemulsion polymerization. Emulsion copolymerization of styr-ene (1.79 g, 18 mmol) and AISA (34 mg, 0.1 mmol) was carriedout in a sealed three-necked round bottom flask equipped witha magnetic stirrer. The surfactant SDS was first dissolved in 45mL of water in the reaction flask. Then the monomers wereadded, and the flask was flushed with nitrogen to removeresidual oxygen. APS (38 mg, 0.17 mmol) was dissolved in 5 mLwater and added dropwise. Polymerization was carried out at70 uC for 24 h. The obtained emulsion was dialysed for 7 days,then vacuum freeze-dried to give PSAISA.
2.5. UV-vis and fluorescence spectral studies
Stock solutions of various biologically relevant ions wereprepared in water. Stock solutions of AISA and PSAISA wereprepared in acetonitrile. The solutions of AISA and PSAISAwere then diluted to the appropriate concentration withphosphate-buffered saline (PBS) (PBS : CH3CN = 1 : 1, v/v,
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pH = 7.4). In the titration experiments, 2 mL of a solution ofAISA and PSAISA was placed in a quartz optical cell of 1 cmoptical path length, and the ion stock solutions were addedgradually using a micropipette. The spectral data wererecorded at different times after the addition of the ions. Forfluorescence measurements, the excitation wavelength was431 nm. The emission and excitation slit widths were both 3.0nm.
2.6. Cell culture and imaging
HeLa cells were cultured in RPMI-1640 supplemented with10% fetal bovine serum (FBS, HyClone) and 1% penicillin–streptomycin in a 5% CO2 atmosphere at 37 uC for 24 h. Forthe H2S detection experiments the cells were first co-incubatedwith PSAISA (40 mg mL21) in RPMI-1640. After incubation for30 min, the cells were washed five times with PBS. Then Na2S(250 mM) was added to the cell samples. After furtherincubation for 5 min, the cells were rinsed three times withPBS and observed with a confocal microscope (Leica, TCSSP5II, Germany).
2.7. Interaction mechanism
A solution of sodium sulfide (43 mg, 0.18 mmol) in 0.4 mLH2O was placed in a stirred solution of AISA (34 mg, 0.1 mmol)in 17 mL acetonitrile. After 2 h reaction at room temperature,the reaction mixture was extracted with chloroform threetimes. After drying with MgSO4, the organic solvents wereevaporated under vacuum. The resulting product was char-acterized by 1H NMR and FT-IR.
3. Results and discussion
3.1. The synthesis of AISA and PSAISA
Monomeric AISA and polymeric PSAISA were synthesized asshown in Scheme 1. 4-Sulfo-1,8-naphthalic anhydride potas-sium salt was used as the starting material. After thesulfonylation and imidization reactions, monomeric AISAwas obtained. The key intermediate compound 2 wassynthesized by the reaction of compound 3, which containsreactive sulfonyl chloride groups, with sodium azide. Since theazide group is bonded directly to the strongly electron-withdrawing sulfonyl group, the sulfonyl azide is rapidlyreduced to sulfonamide by H2S.21 The naphthalic acid imidefluorophore linked to the sulfonyl azide group has severaladvantages such as high fluorescence quantum yield, largeStokes shift and good stability.44 The reduction of sulfonylazide to sulfonamide changes the electronic properties and
thus the fluorescent properties of the naphthalic acid imidemoiety. This can be utilized in the fast, real-time detection ofH2S. The carbon–carbon double bond was introduced intoAISA by an imidization reaction between allylamine andcompound 2. Through the double bond, the monomer canbe further functionalized by polymerization, hydrosilylationand Michael-addition reactions. Moreover, the procedure forthe synthesis of the functional monomer is simple. Themonomer was purified by recrystallization from alcoholwithout column chromatography. The structure was con-firmed by both 1H NMR and FT-IR.
PSAISA was prepared by emulsion polymerization. The datafrom the TEM showed that the PSAISA was in the form ofspherical particles with a diameter of y160 nm (ESI,3 Fig. S4).
3.2. The sensitivity of AISA fluorescence to H2S
Since AISA is the fluorescent component of PSAISA, we firststudied the spectral properties of the monomer AISA. Asshown in Fig. 1, AISA in a PBS–CH3CN solution showed veryweak fluorescence (WF = 0.13). Na2S was used as the hydrogensulfide donor in all experiments because the pKs of H2S (pK1
6.96; pK2 12.90) predict that H2S and HS2 are the predominantsulfide species in aqueous solution whether H2S, NaHS orNa2S is used.40 The fluorescence intensity at 530 nm (emissionmaximum) increased significantly with increasing Na2S con-centration: the addition of 40 mM Na2S resulted in a 5-foldincrease in the fluorescence intensity (inset in Fig. 1). Wespeculate that the fluorescence enhancement of AISA causedby Na2S may be attributed to the reduction of sulfonyl azide tosulfonamide.21 The in vitro detection limit for H2S using AISAwas found to be y2.5 mM. The fluorescence of AISA is clearlyvery sensitive to a change in H2S concentration.
3.3. Specificity and selectivity of AISA for H2S
An important characteristic of any sensor is a good selectivityfor the targeted analyte in a complex, multicomponent system.Therefore we investigated the spectral properties of AISA in thepresence of several biologically relevant anions and cysteine(all having reducing properties). As shown in Fig. 2, highconcentrations of Cl2, Br2, I2, OH2, OAc2, CN2, N3
2, NO22,
HCO32, SO4
22, HSO32, SCN2, S2O3
22 or cysteine led to onlyScheme 1 The synthesis of the functional monomer AISA and the polymericfluorescent PSAISA.
Fig. 1 The fluorescence spectra of AISA (30 mM in PBS–CH3CN) in the presenceof different concentrations of Na2S (0, 5 10, 20, 30 and 40 mM). Inset: therelative responses at 530 nm as a function of Na2S concentration. The data wereacquired 600 s after the addition of Na2S.
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slight changes in the fluorescence intensity whereas a fastfluorescence turn-on was observed with Na2S. These dataindicate that AISA fluorescence is selective for and highlysensitive to H2S.
3.4. The response time of AISA fluorescence to H2S
40 mM Na2S was mixed with 30 mM AISA in PBS–CH3CN (1 : 1,v/v, pH 7.4) and absorption spectra of AISA were taken atdifferent times. As shown in Fig. 3, the absorbance at 373 nmdecreased with reaction time, and a new absorption peakappeared at 431 nm and increased in intensity with time. Thecolorless solution became bright yellow due to the reduction ofsulfonyl azide to sulfonamide.21 The isosbestic point appear-ing at 399 nm suggested the formation of a new substance.35
The effect of reaction time on the fluorescence spectra wasalso studied. As shown in Fig. 4, the fluorescence intensity at530 nm increased with time and reached a maximum at 600 s(inset, Fig. 4). The data indicate that the response of AISA toH2S was fast and the maximum fluorescence intensity could beobtained in a short time. Although faster response times havebeen reported, e.g. 180 s for dansyl azide,21 AISA has otheradvantages. For example, AISA is polymerizable and the
polymeric materials are easily processed for the fabricationof films and optical devices.
3.5. H2S detection for PSAISA
As shown in Fig. 5, the fluorescence spectra of PSAISA in PBS–CH3CN (1 : 1, v/v, pH 7.4) were similar to those of the AISAmonomer. The characteristic fluorescence peak appeared at530 nm and the intensity increased with increasing Na2Sconcentration. The addition of 300 mM Na2S resulted in a3-fold increase in the fluorescence intensity. The fluorescenceenhancement of PSAISA by Na2S was weaker than that of AISA,probably because the polymer chains of the hydrophobicPSAISA are in a random coil conformation in the PBS–CH3CNsolution. Thus the Na2S cannot access all of the AISA residues.Moreover, PSAISA displayed selectivity for hydrogen sulfide(H2S) over other biologically relevant anions and cysteine (ESI,3Fig. S5).
Due to the film-forming properties of polymeric materialsand their ease of processing into a variety of shapes,fluorescent polymers are widely used in the development
Fig. 2 (a) The fluorescence spectra of AISA (30 mM) in PBS–CH3CN (1 : 1, v/v, pH7.4) in the presence of different biologically relevant anions (1 mM Cl2, 1 mMBr2, 1 mM I2, 1 mM OH2, 1 mM OAc2, 1 mM CN2, 1 mM N3
2, 1 mM NO22, 150
mM HCO32, 150 mM HSO3
2, 150 mM SO422, 150 mM S2O3
22, 150 mM SCN2, 40mM Na2S). (b) Samples of AISA under fluorescence (30 mM) in PBS–CH3CN (1 : 1,v/v, pH 7.4) after the addition of 40 mM Na2S, 150 mM SO4
22, 150 mM HSO32,
150 mM S2O322, 150 mM HCO3
2 and cysteine. The data were acquired 600 safter the addition of the anions.
Fig. 3 (a) UV spectra of AISA (30 mM) in PBS–CH3CN (1 : 1, v/v, pH 7.4) in thepresence of 40 mM Na2S at different times (0, 60, 120, 180, 240, 300 and 360 s).(b) Samples of AISA (30 mM) in PBS–CH3CN in the presence of 40 mM Na2S at360 s.
Fig. 4 The fluorescence spectra of AISA (30 mM) in PBS–CH3CN (1 : 1, v/v, pH7.4) in the presence of 40 mM Na2S at different times (0, 60, 120, 180, 240, 300,360, 420, 480, 540, 600, and 660 s). Inset: the relative responses at 530 nm as afunction of reaction time.
Fig. 5 The fluorescence spectra of PSAISA (100 mg mL21) in PBS–CH3CN (1 : 1, v/v, pH 7.4) in the presence of different concentrations of Na2S (0, 40 80, 120, 200and 300 mM). The data were acquired 10 min after the addition of Na2S.
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and manufacture of optical devices. For example, Nishide et al.reported an optical oxygen sensor based on a copolymercoating.45 Tian et al. designed a fluorescent polymer filmsensor for Cu2+ and pyrophosphate anion detection.46
However, to the best of our knowledge, no research onpolymeric fluorescent materials for H2S detection has beenreported so far. The PSAISA material developed in this workprovides a novel approach for the preparation of polymericmaterials for H2S detection.
3.6. Imaging of cells
To further validate the feasibility of the PSAISA sensor materialfor the detection of H2S in vivo, confocal microscope images ofHeLa cells incubated with PSAISA were obtained. As shown inFig. 6, very weak fluorescence was observed in HeLa cells afterincubation with 40 mg mL21 PSAISA for 30 min (Fig. 6b). Thecells were washed five times with PBS and Na2S was added at250 mM; this concentration is well within the range shown toelicit physiological responses (10–600 mM).47,48 After furtherincubation for 5 min, an enhancement of the fluorescence inthe cells was observed (Fig. 6c). Furthermore, a significantincrease in the intracellular fluorescence intensity wasobserved after increasing the reaction time to 15 min(Fig. 6d). Cells have been shown to take up small particles ofless than 200 nm in size, such as mesoporous silicananoparticles, polymers and quantum dots via the endocytosispathway.49,50 In the present work PSAISA spherical particles ofy160 nm in diameter were formed in aqueous solution (ESI,3Fig. S4) and thus could be taken up by the HeLa cells. Afterincubation with Na2S, the intracellular fluorescence intensitywas enhanced by the reduction reaction between Na2S andPSAISA and the intensity increased with an increase of reactiontime due to the formation of more sulfonamide.
3.7. The mechanism of the response of AISA to H2S
To gain a further insight into the optical response of PSAISA toNa2S, AISA and the product of its reaction with Na2S wereexamined by FT-IR and 1H NMR. Since azide groups can be
reduced to amino groups as in organic synthesis, we speculatethat the fluorescence enhancement of AISA caused by Na2Smay be attributed to the reduction of sulfonyl azide tosulfonamide. As shown in Fig. 7A, AISA showed a sharp bandat 2130 cm21, which is assigned to the NMN stretchingvibration. In the presence of Na2S, this band disappeared andtwo new bands appeared at 3458 cm21 and 3342 cm21, whichare assigned to N–H stretching vibrations. The product of theAISA reaction with Na2S was characterized by 1H NMR(Fig. 7B). The appearance of a new peak at 2.0 ppm and itsdisappearance after deuterium exchange clearly indicated theformation of sulfonamide. These IR and 1H NMR data thusconfirmed that the fluorescence enhancement of AISA wascaused by the reduction of sulfonyl azide to sulfonamide in thepresence of Na2S.
4. Conclusions
A novel functional monomer AISA and its copolymer PSAISAwere prepared and showed a selective response to H2S in vitroand in living cells. AISA and PSAISA showed a fast and selectiveH2S-induced fluorescence enhancement. We expect that AISAand PSAISA will be useful for the detection of H2S in bothenvironmental and biomedical applications.
Acknowledgements
The work was financially supported by the National NaturalScience Foundation of China (21174098) and the PriorityAcademic Program Development of Jiangsu Higher EducationInstitutions (PAPD).
Notes and references
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Fig. 6 Confocal microscopy images of HeLa cells. (a) Bright-field images of thefield of cells. (b) Incubated in culture medium containing 40 mg mL21 PSAISA for30 min. (c) Incubated as for (b) followed by the addition of Na2S (250 mM) andincubated for 5 min. (d) After increasing the reaction time to 15 min. The scalebar is 25 mm.
Fig. 7 A: FT-IR spectra of (a) AISA and (b) the product obtained from thereaction of AISA and Na2S. B: 1H NMR spectra: (a) and (b) as in A, (c) after theaddition of D2O to the reaction product. Inset: the proposed mechanism of thereaction of AISA with H2S.
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14548 | RSC Adv., 2013, 3, 14543–14548 This journal is � The Royal Society of Chemistry 2013
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